T-helper cell type 17 lineage-specific adjuvants, compositions and methods

ABSTRACT

The present invention relates to compositions and methods for the modulation of T H 17 responses. The invention provides compositions for the induction of T H 17 responses containing a TLR agonist and an apoptotic cell-associated agent or containing a microbe-infected apoptotic cell. The compositions of the present invention may also contain dendritic cells capable of inducing T H 17 responses. In other embodiments, the invention provides compositions for the inhibition of T H 17 responses containing one or more blocking agents. Methods and compositions for the modulation of T H 17 responses and for the treatment of T H 17-associated diseases and for cancer are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/133,817filed Oct. 3, 2011 which is a U.S. National Phase application under 35 U.S.C. §371 of International Patent Application No. PCT/US2009/067356, filed Dec. 9, 2009, and claims the benefit of U.S. Provisional Patent Application Ser. No. 61/121,449, filed Dec. 10, 2008, all of which are hereby incorporated by reference in their entireties. The International Application published in English on Jun. 17, 2010 as WO 2010/068680 under PCT Article 21(2).

FIELD OF THE INVENTION

The present invention relates to compositions and methods for the modulation of T_(H)17 responses. The invention provides compositions for the induction of T_(H)17 responses containing a TLR agonist and an apoptotic cell-associated agent or containing a microbe-infected apoptotic cell. The compositions of the present invention may also contain dendritic cells capable of inducing T_(H)17 responses. In other embodiments, the invention provides compositions for the inhibition of T_(H)17 responses containing one or more blocking agents. Methods and compositions for the modulation of T_(H)17 responses and for the treatment of T_(H)17-associated diseases and for cancer are also provided

BACKGROUND OF THE INVENTION

The citation and/or discussion of cited references in this section and throughout the specification is provided merely to clarify the description of the present invention and is not an admission that any such references is “prior art” to the present invention.

Adaptive immune responses rely on differentiation of CD4 T helper cells into subsets with distinct effector functions best suited for host defense against an invading pathogen. Interleukin (IL)-17 producing T-helper cells (T_(H)17) are a recently identified subset, separate from the T helper type 1 (T_(H)1) and T helper type 2 (T_(H)2) subsets¹. The T_(H)17 response is a pro-inflammatory T cell response that is associated with the release of IL-17 and IL-23 cytokines. It has been closely associated with immunity in response to TLR-activated microbial infections and has been reported in a variety of autoimmune conditions including inflammatory bowel disease, colitis, systemic sclerosis, psoriasis, rheumatoid arthritis, diabetes, and cystic fibrosis. Studies have shown that inhibitory antibodies to IL-17 or mice deficient in IL-17 responses are able to suppress or prevent the T_(H)17 response in disease models for gastrointestinal immunity, experimental autoimmune encephalitis (EAE), and rheumatoid arthritis.

Generation of T_(H)17 responses is also thought to be favored at mucosal sites for protective immunity. Importantly, mucosal sites represent a primary target for delivery of vaccines, because most infections affect or start from a mucosal surface. Moreover, often in these infections, application of a vaccine directly to the mucosal surface is required to induce an effective, protective immune response [Holmgren, J. and C. Czerkinsky, Nat Med (2005) 11(4 Suppl):S45-53], since induction of peripheral immune responses by parenteral immunization does not result in significant mucosal immunity [Kiyono, H., et al. Reg Immunol, (1992) 4(2):54-62; McGhee, J. R., et al., Vaccine, (1992) 10(2):75-88].

The effort that has been focused thus far on inducing effective immune responses in mucosal tissues has met with considerable challenge, since most protein antigens (Ags) are rather weak immunogens when given via the mucosal route. The coadministration of mucosal adjuvants, such as cholera toxin (CT), has been shown to effectively support Ag-specific mucosal immune responses, however, the inherent toxicity of cholera toxin is a major hindrance to its use in humans. Thus, the development of other, effective and reliable mucosal adjuvants, that preferably induce T_(H)17 responses, is of central importance for new generation vaccines [McGhee, J. R., et al. Vaccine (1992) 10(2)75-88]. However, it is important to recognize that tight regulation of these T_(H)17 responses must also be achieved, since T_(H)17 responses are also associated with driving a number of autoimmune diseases. Thus, it is critical that the factors that govern differentiation of T_(H)17 cells in vitro be better understood, so that T_(H)17 responses may be carefully regulated.

SUMMARY OF THE INVENTION

In certain embodiments of the present invention, a microbe-infected apoptotic cell that expresses an exogenous immune antigen is provided.

In other embodiments of the present invention an isolated T_(H)17-inducing dendritic cell (DC) that secretes interleukin-6 (IL-6) and transforming growth factor beta (TGF-β), is provided, wherein the combined amount of IL-6 and TGF-β is effective for inducting a T_(H)17 response. In certain embodiments, the present invention provides an isolated T_(H)17-inducing dendritic cell (DC) wherein, the T_(H)17-inducing DC is loaded with an apoptotic cell which includes a TLR ligand or an inactivated microbe, and wherein the microbe expresses an exogenous immune antigen. In still other embodiments, the present invention provides a composition including a T_(H)17-inducing dendritic cell (DC) and interleukin-6, wherein the DC is loaded with an apoptotic cell, and wherein the apoptotic cell expresses an exogenous immune antigen.

In certain aspects of the present invention, a method for inducing a T_(H)17 response in a mammal is provided, which involves administering to a mammal in need of such induction a microbe-infected apoptotic cell of the invention is an effective amount for inducing the T_(H)17 response.

In other aspects, the present invention provides a method for inducing a T_(H)17 response in a mammal, which involves administering to a mammal in need of such induction a T_(H)17-inducing DC of the invention in an effective amount for inducing the T_(H)17 response. In certain aspects, the method further involves administering to a DC in vitro a microbe-infected apoptotic cell in an effective amount for generating a T_(H)17-inducing DC of the invention. In yet other aspects, the microbe-infected apoptotic cell expresses an exogenous immune antigen.

In certain embodiments, a method for generating the T_(H)17-inducing DC of the invention involves administering to a DC in vitro a Toll-like receptor (TLR) agonist and an apoptotic cell-associated agent in a combined amount effective for generating the T_(H)17-inducing DC. In some embodiments, the TLR agonist and the apoptotic cell-associated agent are either in direct physical association or are combined in a manner that allows internalization as a single entity by the DC in vitro. In still other embodiments of the invention, methods of generating a TH17-inducing DC involve administering to a DC in vitro, as a single entity or in a combined form, at least one member selected from the group consisting of a Toll-like receptor (TLR) ligand, a TLR ligand mimic, a synthetic or chemical TLR ligand, a cell or particle including a pathogen-associated molecular pattern, a microbial pathogen, a TLR agonist, a bacterium, and a virus or viral-like particle, and at least one member selected from the group consisting of an apoptotic cell, a microbe-infected apoptotic cell, an apoptotic cell mimic, phosphatidylserine, a phosphatidylserine mimic, an apoptotic cell-associated agent, a mimic of cell surface calreticulin translocation, and a polypeptide that is a marker of apoptosis, in a combined amount effective for generating the T_(H)17-inducing DC.

In certain aspects of the present invention, a vaccine composition is provided which includes: a) a microbe-infected apoptotic cell, b) an immune antigen, and c) a pharmaceutically acceptable carrier or diluent, wherein the combined amount of a) and b) is effective for eliciting an immune response directed toward the immune antigen. In some aspects, the microbe-infected apoptotic cell expresses the immune antigen, and the immune antigen is an exogenous immune antigen.

In some embodiments of the present invention, a vaccine composition is provided that includes: a) a T_(H)17-inducing dendritic cell (DC) that secretes interleukin-6 (IL-6) and transforming growth factor beta isoform 1 (TGF-62 ), b) an immune antigen, and c) a pharmaceutically acceptable carrier or diluent, wherein a combined amount of IL-6 and TGF-β secreted by the DC and the immune antigen is effective for eliciting a T_(H)17 response to the immune antigen. In certain embodiments, methods for generating the T_(H)17-inducing DC include pre-treating a DC in vitro with a Toll-like receptor (TLR) agonist and an apoptotic cell-associated agent in a combined amount effective for generating the T_(H)17-inducing DC. In other embodiments, the TLR agonist and the apoptotic cell-associated agent are combined in a manner that allows internalization as a single entity by the DC in vitro. In still other embodiments, the DC is treated with the apoptotic cell-associated agent and administered with recombinant interleukin-6 (IL-6). In yet other embodiments, the T_(H)17-inducing DC is pre-treated in vitro with the immune antigen or with a peptide fragment derived from the immune antigen.

In certain embodiments, vaccine compositions provided by the present invention further include a microbe-infected apoptotic cell, and a combined amount of the microbe-infected apoptotic cell, a) and b) is effective for eliciting an immune response.

In certain aspects of the present invention, methods for treating of preventing cancer in a mammal are provided, which involve administering to a mammal in need of such treatment a vaccine composition of the invention in an effective amount for treating or preventing cancer, wherein the antigen is a tumor-specific antigen. In some aspects, the cancer is an epithelial or mixed epithelial carcinoma. In certain of these aspects, the epithelial or mixed epithelial carcinoma is a member selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, lung carcinoma, laryngeal carcinoma, adenoid cystic carcinoma, epithelial carcinomas of the upper aerodigestive tract, hepatocellular carcinoma, colorectal carcinoma, lymphoepithelial carcinoma, squamous cell carcinoma, renal cell carcinoma, mixed epithelial and stromal tumors of the kidney, and renal angiomyoadenomatous tumors.

In other aspects, the present invention provides a method for inducing in a patient a T_(H)17-driven immune response to an antigen, which involves administering to a patient in need of such treatment the vaccine composition according to certain of the above aspects of the present invention in an effective amount for inducing a T_(H)17-driven immune response.

In certain aspects of the present invention, a method is provided for modulating an immune response of a mammal, which involves administering to a mammal in need of such treatment a vaccine composition according to certain of the above aspects of the present invention in an effective amount for modulating the immune response of the mammal.

In some embodiments of the present invention, a method for inhibiting a T_(H)17 response in a mammal is provided, which involves administering to a mammal in need of such treatment a blocking agent that inhibits immune recognition of an apoptotic cell-associated agent in an effective amount for inhibiting the T_(H)17 response in the mammal. In yet other embodiments, a method for inhibiting a T_(H)17 response in a mammal, involves administering to a mammal in need of such treatment a blocking agent that inhibits immune recognition of a Toll-like receptor (TLR) adjuvant in an effective amount for inhibiting the T_(H)17 response in the mammal.

In certain embodiments of the invention, a method for inducing regulatory T cell development and immune tolerance in a mammal is provided, which involves administering to a mammal in need of such treatment a blocking agent that inhibits immune recognition of a Toll-like receptor (TLR) ligand in an effective amount for inducing the regulatory T cell development and immune tolerance in the mammal, wherein the TLR ligand is a component of an infected apoptotic cell.

In yet other embodiments, a method for inducing immune tolerance in a mammal is provided, which involves administering to a mammal in need of such treatment an apoptotic cell-associated agent in an effective amount for inducing regulatory T cell development and immune tolerance in the mammal. In still other embodiments, a method for inducing immune tolerance in a mammal is provided, which involves administering to a mammal in need of such treatment a blocking agent that inhibits immune recognition of a Toll-like receptor (TLR) adjuvant in an effect amount for inducing immune tolerance in the mammal.

In certain aspects, the present invention provides a methods for inducing immune tolerance in a mammal, which involves administering to a mammal in need of such treatment a blocking agent that inhibits immune recognition of a Toll-like receptor (TLR) adjuvant or blocks TLR signal transduction in an effective amount for inducing immune tolerance in the mammal.

In one embodiment of the present invention, a composition is provided having a first blocking agent that inhibits immune recognition of an apoptotic cell-associated agent, and a second blocking agent that inhibits immune recognition of a Toll-like receptor (TLR) adjuvant.

In yet other embodiments, a pharmaceutical formulation is provided including a first clocking agent that inhibits immune recognition of an apoptotic cell-associated agent, and a second clocking agent that inhibits immune recognition of a Toll-like receptor (TLR) adjuvant, and pharmaceutically acceptable diluent or carrier. In certain of these embodiments, the first blocking agent specifically inhibits dendritic-cell-mediated immune recognition of the apoptotic cell-associated agent. In other embodiments, the second blocking agent specifically inhibits dendritic-cell-mediated immune recognition of the TLR adjuvant.

The present invention provides in some embodiments, a method for the treatment of a T_(H)17-driven disease or condition in a mammal, which involves administering to a mammal in need of such treatment a pharmaceutical formulation of the invention in an effective amount for treating the T_(H)17-driven disease or condition, wherein the disease or condition is a member selected from the group consisting of inflammatory bowel disease. Crohn's disease, colitis, systemic sclerosis (scleroderma), atopic dermatitis, psoriasis, rheumatoid arthritis, diabetes, cystic fibrosis, allergic airway disease, atopic asthma, allergic asthma, Sjogren's Syndrome, and systemic lupus erythematosus.

In certain aspects, the invention provides a vaccine composition including: a) a first quantity of a blocking agent which inhibits immune recognition of an apoptotic cell-associated agent, b) a second quantity of a blocking agent which inhibits immune recognition of Toll-like receptor (TLR) adjuvant, c) a third quantity of an immune antigen, and d) a pharmaceutically acceptable carrier or diluent, wherein the combined quantities of a), b) and c) are effective for inhibiting a T_(H)17 response. In certain aspects, the combined quantities of a), b) and c) are effective for inducing a T regulatory cell response. In other aspects, the immune antigen is a rumor-specific antigen.

The present invention provides, in certain embodiments, a method for treating or preventing cancer in a subject, which involves administering to a subject in need of such treatment a vaccine composition of the present invention in an effective amount for treating or preventing cancer, wherein the antigen is a tumor-specific antigen. In certain of these embodiments, the subject is a human or mammal. In other aspects, the human is a patient. In other aspects, the cancer is a member selected from the group consisting of Hodgkin lymphoma, follicular lymphoma, multiple myeloma, monoclonal gammopathy and T cell leukemia/lymphoma.

In some aspect of the invention, a method for treatment of T_(H)17-driven disease or condition is provided, which involves administering to a mammal in need of such treatment a vaccine composition according to the present invention in a effective amount for treating a T_(H)17-driven disease that is a member selected from the group consisting of inflammatory bowel disease, Crohn's disease, colitis, systemic sclerosis (scleroderma), atopic dermatitis, psoriasis, rheumatoid arthritis, diabetes, cystic fibrosis, allergic airway disease, atopic asthma, allergic asthma, Sjogren's Syndrome, and systemic lupus erythematosus.

In any of the above embodiments of the invention, the apoptotic cell-associated agent includes any one of the agents selected from the group consisting of an apoptotic cell, an apoptotic cell mimic, phosphatidylserine, a microbe-infected apoptotic cell, a phosphatidylserine mimic, a mimic of cell surface calreticulin translocation, and a polypeptide that is a marker of apoptosis.

In any of the above embodiments of the invention, the TLR adjuvant includes any one of the agents selected from the group consisting of a TLR ligand, a TLR ligand mimic, a synthetic or chemical TLR ligand, a cell or particle including a pathogen-associated molecular pattern, a microbial pathogen, a bacterium, and a virus or viral particle.

In any of the above aspects of the invention, the mammal may be a human and a patient may be a human.

In any of the above embodiments of the invention, a microbe may be selected from the group consisting of attenuated live Mycobacterium bovis, Salmonella typhi, and Vibrio cholerae.

In any of the above embodiments of the invention, the vaccine composition is delivered by an oral or mucosal route.

In any of the above aspects of the invention, the T_(H)17 or immune response is a mucosal immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows protein levels of cytokines produced by bone marrow dendritic cells following in vitro stimulation with LPS, apoptotic A20/LPS blasts, or apoptotic neutrophils/E. coli.

FIG. 2 shows mRNA levels of cytokines produced by bone marrow dendritic cells following in vitro stimulation with LPS, apoptotic A20/LPS blasts, or apoptotic neutrophils/E. coli.

FIG. 3 shows protein levels of IL-17 produced by CD4 T cells following culture with LPS or TGF-β+IL-6, or following culture with conditioned media from dendritic cells that phagocytosed apoptotic B cells, apoptotic cells+IL-6, or apoptotic B cell LPS blasts.

FIG. 4 shows mRNA expression levels of Rorγt (Rorc), IL-17A, IL-22, T-bet, Foxp3, and IL-10 in CD4 T cells following culture with LPS or TGF-β+IL-6, or following culture with conditioned media from dendritic cells that phagocytosed apoptotic B cells, apoptotic cells+IL-6, or apoptotic B cell LPS blasts.

FIG. 5 shows FACS plots of intracellular cytokine staining of Foxp3, IL-17, and IFN-γ in CD4 T cells following culture with conditioned media from dendritic cells that phagocytosed the indicated stimuli.

FIG. 6 shows FACS plots of intracellular cytokine staining of CD4 and Foxp3 or antibody isotype control in CD4 T cells following culture with conditioned media from dendritic cells isolated from C57BL/6J (B6), MyD88^(−/−)/TRIF^(−/−), or TLR4^(−/−) mice, that had phagocytosed the indicated stimuli.

FIG. 7 shows FACS plots of intracellular cytokine staining of IFN-γ and IL-17 in CD4 T cells following culture with conditioned media from dendritic cells that had phagocytosed the indicate stimuli.

FIG. 8 shows protein levels of IL-17 in the culture supernatants of CD4 T cells following culture with conditioned media from dendritic cells that had phagocytosed apoptotic LPS blasts and in the presence of absence of the indicated TGF-β, p19, or IL-6 neutralizing antibodies.

FIG. 9 shows protein levels of IL-17 in the culture supernatants of CD4 T cells following culture with the indicated cytokines only, or following culture with conditioned media from C57BL/6J (WT) or IL-6^(−/−) dendritic cells that had phagocytosed the indicated stimuli.

FIG. 10 shows protein levels of IL-6 and IL-12 in the culture supernatants of CD4 cells following culture with conditioned media from C57BL/6J (WT) or IL-6^(−/−) dendritic cells that had been treated with LPS or that had phagocytosed the indicated stimuli.

FIG. 11 shows protein levels of IL-17 in the culture supernatants of CD4 T cells following culture in the presence of LPS, curdlan, or curdlan+TGF-β, or following culture with conditioned media from C57BL/6J (WT) or MyD88^(−/−) TRIF^(−/−) dendritic cells that had phagocytosed the indicated stimuli.

FIG. 12 shows protein levels of IL-17 in the culture supernatants of CD4 T cells following culture in the presence of LPS, curdlan, or curdlan+TGF-β, or following culture with conditioned media from C57BL/6J (WT) or TLR4^(−/−−) dendritic cells that had phagocytosed the indicated stimuli.

FIG. 13 shows FACS plots of intracellular cytokine staining of IL-17 and IL-10 in CD4 T cells culture with TGF-β, TGF-β+IL-6, or LPS, or following culture with conditioned media from dendritic cells that had phagocytosed the indicated stimuli.

FIG. 14 shows the protein levels of IL-17 and IL-10 in the culture supernatants of CD4 T cells cultured in the presence or absence of IL-23, that had also been cultured with IL-6, TGF-β, TGF-β+IL-6, or LPS, or with conditioned media from dendritic cells that had phagocytosed the indicated stimuli.

FIG. 15 shows the protein level of IL-10 in the culture supernatants of CD4 T cells following culture in the presence of LPS, curdlan, or curdlan+TGF-β, or following culture with conditioned media from C57BL/6J (WT) or MyD88^(−/−) TRIF^(−/−) dendritic cells that had phagocytosed the indicated stimuli.

FIG. 16 shows the protein level of IL-10 in the culture supernatants of CD4 T cells following culture in the presence of LPS, curdlan, or curdlan+TFGF-β, or following culture with conditioned media from C57BL/6J (WT or TLR4^(−/−) dendritic cells that had phagocytosed the indicated stimuli.

FIG. 17 shows FACS plots of intracellular cytokine staining of IL-10, IL-17, and IFN-γ in CD4 T cells following culture with conditioned media from C57BL/6J (B6) or MyD88^(−/−) TRIF^(−/−) dendritic cells that had been treated with LPS or that had phaocytosed the indicated stimuli.

FIG. 18 is a FACS plot of 7AAD- and Annexin V-stained apoptotic A20 B cells (treated with anti-Fas) showing that Q-VD-OPH inhibits apoptosis.

FIG. 19 is a graph quantifying the amount of TUNEL staining in sections of colonic epithelial cells taken from the colons of mice infected with the indicated bacteria in the presence or absence of the caspase inhibitor Q-VD-OPH.

FIG. 20 shows FACS plots of intracellular cytokine staining of CD4, CD8α, IFN-γ, and IL-17 in cells isolated from the indicated tissues from mice infected with the indicated bacteria or treated with DSS.

FIG. 21 shows FACS plots of intracellular cytokine staining of IFN-γ and IL-17 in cells isolated from the indicated tissues from mice infected with the indicated bacteria.

FIG. 22 shows FACS plots of intracellular cytokine staining if IFN-γ and IL-17 in cells isolated from the indicated tissues from mice treated with DSS.

FIG. 23 shows FACS plots of intracellular cytokine staining of CD4, CD8α, IFN-γ, and IL-17 in cells isolated from the indicated tissues from mice infected with Citrobacter rodentium.

FIG. 24 shows FACS plots of intracellular cytokine staining in CD4 T cells following culture with condition medium.

FIG. 25 shows FACS plots of intracellular cytokine staining of CD4 T cells following culture with conditioned medium from DC that were given apoptotic cells and soluble LPS separately (Apoptotic A20+LPS) or from DC that were given apoptotic cells with the TLR ligand physically integrated (Apoptotic A20/LPS blasts).

DETAILED DESCRIPTION

The present invention relates to the surprising discovery of naturally-occurring, specific combination of immune adjuvants that induces a T_(H)17 response in vitro and in vivo. More specifically, the present invention provides methods for regulating T_(H)17 responses based on this discovery. In certain embodiments, the compositions of the present invention are useful for inducing T_(H)17 responses. In other embodiments, the compositions of the invention are useful for inhibiting T_(H)17 responses and for inducing immune tolerance.

Also provided by the present invention are pharmaceutical formulations and vaccine compositions useful for modulating T_(H)17 responses.

Characteristics of CD4 T Cell Subsets

CD4 T cells upon activation and expansion develop into different T helper (T_(H)) cell subsets with different cytokines profiles and distinct effector functions. Appropriate differentiation of T_(H) into effector subsets best suited for host defense against an invading pathogen is of critical importance to the immune system. CD4 T cells differentiate into at least four known subsets, three effector subsets (T_(H)1, T_(H)2 and T_(H)17) and one T regulatory subset (T_(reg)).

Based on the cytokines that they produce, T cells were historically divided into T_(H)1 and T_(H)2 cells, and this has provided a framework to understand how specific cytokine milieus produced by cells of the innate immune system guide the development of adaptive immunity. T_(H)1 cells, which are potently induced by dendritic cells (DC) secreting IL-12, are characterized by the expression of the lineage-specific transcription factor T-bet (T box 21) and the production of IFN-γ. T_(H)2 cells, which depends on IL-4 during differentiation and lack of IL-12, produce IL-4, IL-5, IL-9, and IL-13 and are characterized by the expression of the transcription factor GATA-3. Importantly, in the past five years, a third subset of IL-17-producing effector T helper cells, called T_(H)17 cells, has been discovered and characterized.

T_(H)17 cells produce IL-17, IL-17F and IL-22. By secreting these effector cytokines, T_(H)17 cells induce a massive tissue reaction due to the broad distribution of the IL-17 and IL-22 receptors. T_(H)17 cells also secrete IL-21 to communicate with the cells of the immune system. Synergy between the cytokines transforming growth factor beta isoform 1 (TGF-β) and interleukin (IL)-6 induces development of T_(H)17 cells in mice¹⁰⁻¹² and humans¹³, while IL-23 supports expansion of these cells¹⁰⁻¹². IL-23 consists of two subunits, the p19 subunit and the p35 subunit. The p35 subunit is also used by IL-12, which is a heterodimer consisting of the p35 and the p40 subunit. The differentiation factors (TGF-βplus IL-6 or IL-21), the growth and stabilization factor (IL-23), and the transcription factors (STAT3, ROR-γt (ROR-c), and ROR-a) involved in the development of T_(H)17 cells have only recently been identified. The participation of TGF-β in the differentiation of T_(H)17 cells places the T_(H)17 lineage in close relationship with CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (T_(reg)) since TGF-β also induces differentiation of naive T cells in Foxp3⁺ T_(reg) in the peripheral immune compartment. The investigation of the differentiation, effector function, and regulation of T_(H)17 cells has opened up a new framework for understanding T cell differentiation. While the important of T_(H)17 cells in clearing pathogens during host defense reaction and in inducing tissue inflammations in autoimmune disease has been appreciated [Reviewed in Kota, T. et al. (2009) Annual Review of Immunology, 27:485-517], the exact nature of the stimuli that induce their differentiation in vivo is not known.

T_(reg) cells are specialized subpopulation of T cells that act to suppress motivation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Development of T_(reg) cells, which are capable of suppressing autoimmune disease, is therefore reciprocally related to T_(H)17 cells,¹⁰ which can drive immune responses, including autoimmune responses. T_(reg) cells can be identified by their unique expression of the transcription factor forkhead box P3 (Foxp3).¹⁹ Importantly, there are two phenotypically identical populations of CD4⁺CD25⁺ T_(reg)—natural and adaptive. Natural CD4⁺CD25⁺ T_(reg) cells arise in the thymus under homeostatic conditions to safeguard against autoimmunity. Adaptive CD4⁺CD25⁺ T_(reg) cells arise during inflammatory processes such as infections and cancers and suppress immunity through heterogeneous mechanisms that include direct contact or the production of soluble factors such as IL-10 and TGF-β.

T_(reg) cells are thought to be involved in cancer. The tumor itself and cells in the tumor microenvironment, such as DC, induce the differentiation of T_(reg) cells through various mechanisms including the production of TGF-β and the expression of the costimulatory molecule B7-H1. At least some tumor-associated T_(reg) cells are specific for tumor antigens, although once activated, they can also suppress tumor antigen-independent immune responses through bystander mechanisms. It has been demonstrated that T_(reg) cells actively accumulate in the human ovarian cancer microenvironment, inhibiting tumor-specific cytotoxicity and cytokine production by tumor-specific CD8⁺ T cells in vitro and in vivo. Importantly, certain studies have shown that the number of CD4⁺CD25⁺Foxp3⁺ T cells correlates inversely with clinical outcomes in several epithelial carcinomas, including ovarian cancer, breast cancer, and hepatocellular carcinoma. [Curiel et al. (2007) Clin Invest. 117(5):1167-1174]. In one study, there was an inverse correlation between the number of T_(reg) cells in the tumor and patient survival, which was corroborated independently by the demonstration that high levels of Foxp3 in the tumor microenvironment predicted reduced survival in patients with ovarian cancer. Thus, in ovarian cancer, for example, it would be useful to be able to inhibit development of T_(reg) responses, for example, through the induction of T_(H)17 responses. The present invention provides such methods.

While the presence of T_(reg) cells is thought to be harmful in some cancers, as discussed above, in others it may be helpful. For example, the situation in hematologic malignancies differs. Increased numbers of tumor-infiltrating Foxp3⁺ T cells predicts improved survival in individuals with follicular lymphoma, and reduced numbers of Foxp3⁺ cells predicts poor survival in individuals with Hodgkin lymphoma. Both the number and the functions of Foxp3⁺ T_(reg) cells was reduced in patients with multiple myeloma or monoclonal gammopathy of uncertain significance, and T_(reg) cell function decreased as tumor burden increased in a small series of patients with cutaneous T cell leukemia/lymphoma. Therefore, it is possible that the effects of T_(reg) cells or the differentiation of Foxp3⁺ cells in functional T_(reg) cells fundamentally differs in lymphoid malignancies compared with epithelial carcinomas. These studies indicate that in certain circumstances, it would be useful to be able to induce T_(reg) cell responses, likely through the modulation of T_(H)17 responses, in order to treat cancer. The present invention provides such methods.

In certain embodiments, the epithelial or mixed epithelial carcinomas that may be treated by methods of the present invention include ovarian cancer, breast cancer, pancreatic cancer, lung carcinoma, laryngeal carcinoma, adenoid cystic carcinoma, epithelial carcinomas of the upper aerodigestive tract, hepatocellular carcinoma, colorectal carcinoma, lymphoepithelial carcinoma, squamous cell carcinoma, renal cell carcinoma, mixed epithelial and stromal tumors of the kidney, and renal angiomyoadenomatous tumors.

Generation of T_(H)17 Cells

T_(H)17 cells have been known to be induced in vitro by TGF-β and IL6¹⁻⁵. However, it was not known what conditions in vivo would induce this combination of cytokines. Furthermore, it is enigmatic that a combination of pro-inflammatory and anti-inflammatory cytokines would be required to generate an effector T_(H)17 response. The present invention shows that the relevant physiological stimulus triggering this combination of T_(H)17-inducing cytokines is the recognition and phagocytosis of infected apoptotic cells by DC. Phagocytosis of infected apoptotic cells uniquely triggers the combination of IL-6 and TGF-β through recognition of a pathogen associated molecular pattern (PAMP) (e.g., Toll-like receptor (TLR) ligands)⁶ and an apoptotic-cell associated agent (such as, e.g., phosphatidylserine exposed on apoptotic cells⁷), respectively. Conversely, phagocytosis of apoptotic cells in the absence of microbial signals induces differentiation of the closely related regulatory T-cells (T_(reg)), which are important for controlling autoimmunity⁸.

A surprising discovery of the present invention was that the TLR ligand (or other PAMP) and the apoptotic-cell associated agent must be associated as a single entity (e.g., as a microbe-infected apoptotic cell) in order to generate a T_(H)17-inducing DC. Coadministration of the TLR ligand and apoptotic cell-associated agent did not generate a T_(H)17-inducing DC. Moreover, blocking apoptosis during infection of the intestinal epithelium with the rodent pathogen Citrobacter rodentium ⁹, impaired the characteristic T_(H)17 response in the lamina propria. The results of the present disclosure demonstrate that infected apoptotic cells are a critical component of the innate immune signals instruction T_(H)17 differentiation, and point to pathogens particularly adept at triggering apoptosis that might preferentially induce T_(H)17-mediated immunity.

The coding sequences and amino acid sequences for human and murine IL-6, IL-10, IL-12 p35 and p40 subunits, IL-22, IL-23a p19 subunit, TGF-β1, Foxp3, TNFsf15, IL-17A, IL-17F, Tbet (TBX21), Ror-c (Ror-γt), β-actin, and HPRT are known and have been described. The coding sequences are set forth in the sequence identifiers as follows: hIL-6 (NM_(—)000600) (SEQ ID No: 1), mIL6 (NM_(—)031168) (SEQ ID NO: 2), hIL10 (NM_(—)000572) (SEQ ID NO: 3), mIL10 (NM_(—)010548) (SEQ ID NO: 4), hIL-12a(p35 subunit) (NM_(—)000882) (SEQ ID NO: 5), hIL-12b (p40 subunit) (NM_(—)002187) (SEQ ID NO: 6), mIL-12a p35 subunit (NM_(—)008351) (SEQ ID NO: 7), mIL-12b p40 subunit (NM_(—)008352) (SEQ ID NO: 8), hIL-22 (NM_(—)020525) (SEQ ID NO: 9), mIL-22 (NM_(—)016971) (SEQ ID NO: 10), hIL-23a p19 subunit (NM_(—)016584) (SEQ ID NO: 11), mIL-23a p19 subunit (NM_(—)031252) (SEQ ID NO: 12), hTGFβ (NM_(—)000660) (SEQ ID NO: 13), mTGFβ (NM_(—)011577) (SEQ ID NO: 14), hFoxp3 (NM_(—)014009) (SEQ ID NO: 15), mFoxp3 (NM_(—)054039) (SEQ ID NO: 16), hTMFsf15 (NM_(—)005118) (SEQ ID NO: 17), nTNFsf15 (NM_(—)177371) (SEQ ID NO: 18), hIL-17A (NM_(—)002190) (SEQ ID NO: 19), mIL-17A (NM_(—)010552) (SEQ ID NO: 20), hIL-17F (NM_(—)052872) (SEQ ID NO: 23), mIL-17F (NM_(—)145856) (SEQ ID NO: 24), hTbet (TBX21) (NM_(—)013351) (SEQ ID NO: 25), mTbet (Tbx21) (NM_(—)019507) (SEQ ID NO: 26), hRORc (NM_(—)005060) (SEQ ID NO: 27), mRORc (NM_(—)011281) (SEQ ID NO: 28), hHPRT (NM_(—)000194) (SEQ ID NO: 29), mHPRT (NM_(—)013556) SEQ ID NO: 30), hβ-actin (NM_(—)001101) (SEQ ID NO: 31), and mβ-actin (NM_(—)007393) (SEQ ID NO: 32).

The amino acid sequences are set forth in the sequence identifiers as follows: hIL-6 (NP_(—)000591) (SEQ ID NO: 33), mIL6 (NP_(—)112445) (SEQ ID NO: 34), hIL10 (NP_(—)000563) (SEQ ID NO: 35), mIL10 (NP_034678) (SEQ ID NO: 36), hIL-12a(p35 subunit) (NP_(—)00873) (SEQ ID NO: 37), hIL-12b (p40 subunit) (NP_(—)002178) (SEQ ID NO: 38), mIL-12a p35 subunit (NP_032377) (SEQ ID NO: 39), mIL-12b p40 subunit (NP_(—)032378) (SEQ ID NO: 40), hIL-22 (NP_065386) (SEQ ID NO: 41), mIL-22 (NP_(—)058667) (SEQ ID NO: 42), hIL-23a p19 subunit (NP_(—)057668) (SEQ IS NO: 92), mIL-23a p19 subunit (NP_(—)112542) (SEQ ID NO: 93), hTGFβ (NP_(—)000651) (SEQ ID NO: 43), mTGFβ (NP_(—)035707) (SEQ ID NO: 44), hFox3 (NP_(—)054728) (SEQ ID NO: 45), mFoxp3 (NP_(—)473380) (SEQ ID NO: 46), hTNFsf15 (NP_(—)005109) (SEQ ID NO: 47), mTNFsf15 (NP_(—)796345) (SEQ ID NO: 48), hIL-17A (NP_(—)002181) (SE ID NO: 49), mIL-17A (NP_(—)034682) (SEQ ID NO: 50), hIL-17F (NP_(—)443104) (SEQ ID NO: 51), mIL-17F (NP_(—)665855) (SEQ ID NO: 52, hTbet (TBX21) (NP_(—)037483) (SEQ ID NO: 53), mTbet (Tbx21) (NP_(—)062380) (SEQ ID NO: 54), hRORc (RORγτ) (NP_(—)005051) (SEQ ID NO: 55), mRORc (RORγτ) (NP_(—)035411) (SEQ ID NO: 56), hHPRT (NP_(—)000185) (SEQ ID NO: 57), mHPRT (NP_(—)038584) (SEQ ID NO: 58), hβ-actin (NP_(—)001092) (SEQ ID NO: 59), and mβ-actin (NP_(—)031419) (SEQ ID NO: 60).

Compositions and Methods for Inducing a T_(H)17 Response in a Mammal

In certain embodiments, the invention relates to compositions for inducing T_(H)17 responses which include a Toll-like receptor (TLR) agonist. The innate immune system in mammals senses the invasion of microorganisms using pattern recognition receptors (PRRs), such as the family of TLRs, which recognize conserved microbial components, termed pathogen-associated molecular patterns (PAMPs). Activation of TLRs leads to the induction of inflammatory responses and the development of antigen-specific adaptive immunity. [Reviewed in Takeda, K. et al. (2003) Annual Review of Immunology, 21:335-376]. TLRs are characterized by an extracellular domain composed of leucine-rich-repeat motifs for ligand binding as well as an IL-1 receptor domain (termed TIR domain). TLR intracellular domains specifically recruit several adaptor proteins including MyD88, TRIF, TIRAP/MAL, TOLLIP, and/or TRAM for downstream signaling. These adaptor proteins subsequently associate with a family of IL-1 receptor associated kinases (IRAK1, 2, M, and 4). Recruitment of numerous downstream signaling proteins lends to activation of a range of transcription factor such as NF-κB, AP-1, and IRFs, which are responsible for specific gene transcription, including the genes for pro-inflammatory cytokines including IL-6, IL-10, IL-17, and TGF-β. Despite significant domain conservation, distinct TLRs or combinations of TLR heterodimers induce gene programs that lead not only to the robust production of general proinflammatory mediators but also to the production of unique effectors, which provide pathogen-tailored immune responses.

Biochemical studies and genetic analyses using transgenic mice have revealed specific ligands for the activation of TLRs. Of the 11 TLRs described, the ligands for 10 of the receptors have been identified. TLR1, TLR2, TLR4 and TLR6 (both as heterodimers and homodimers) recognize different microbial structures, whereas TLR3 recognizes viral double stranded RNA (dsRNA). TLR5 recognizes Flagellin, a protein found in the flagella of gram-negative bacteria. TLR7 and 8 recognize endosomal single-stranded RNA (ssRNA) to detect infection by virus, and TLR9 detects unmethylated CpG motifs, characteristics of bacterial DNA. TLR11, present in mice, but not humans, senses the profilin-like proteins from the protozoan parasite Toxoplasma gondii and also recognizes uropathogenic E. coli.

In certain embodiments of the invention, the requirement for TLR signaling for the generation of a response to an antigen or microbial pathogen or product is tested in vivo using genetically engineered “knockout” mice. For example, TLR4 knockout (TLR4^(−/−)) mice are homozygous null for the full-length TLR4 gene. Accordingly, these mice do not express functional TLR4 protein and therefore cannot detect TLR4 ligands, such as, e.g., lipopolysaccharide (LPS) through TLR4. In other knockout mice, adaptor molecules that are downstream of TLRs in the signaling pathway are targeted for deletion. For example, MyD88^(−/−) mice do not express the MyD88 adaptor protein, and are deficient in most TLR signaling pathways. It has been discovered the some TLRs do not depend entirely, or at all, on MyD88, however, because the signal is transmitted through another adaptor molecule, TRIF. Thus, TRIF^(31 /−) mice have been generated to determine which TLRs depend on TRIF for signaling (such as, e.g., TLR3). It has also been shown that when MyD88^(−/−) TRIF^(−/−) double knockout mice are used (i.e., they express neither functional MyD88 nor TRIF proteins), all TLR signaling is completely abrogated. [See, Kawai T and Akira S. (2007) Semin Immunol; 19:24-32; Medzhitov R. (2007) Nature; 449:819-26].

TLRs are expressed on a wide variety of cells in mammals, including on cells of the innate (such as antigen-presenting cells (APC)) and adaptive immune systems. The DC is an APC that is of critical importance for the initiation of adaptive immune responses. DC reside systemically, in lymph nodes, and in tissues, where they are poised for early detection of microbial invasion, and to signal the invasion to the adaptive arm of the immune system (i.e., to T and B cells). However, DC reside in an immature state, and in this state they are unable to stimulate T cell activation. It is through recognition of microbial pathogens or products by PRRs (especially TLRs), expressed on their cell surface or intracellularly, that activation of these cells is facilitated (upon, e.g., phagocytosis of the invading pathogen or an associated antigens). Phagocytosis of apoptotic cells by DC is described in detail in Blander, J. M. and Medzhitov., R, Science (2004) 304 (5673):1014.

Upon TLR ligation, DC undergo a program of maturation whereby they upregulate costimulatory molecules (e.g., CD80, CD86, CD40 and major histocompatibility complex (MHC) type I and type II molecules), which are critical for T cell activation, and they migrate to the draining lymph node associated with the assaulted tissue. In the draining lymph node, the mature DC present peptides from the phagocytosed pathogen in the context of surface MHC molecules to naïve T cells in the draining lymph node, leading to T cell activation. “Naïve T cells” are T cells which have never encountered the antigen for which their unique T cell receptor is specific.

Importantly, in the draining lymph nodes, mature DC secrete cytokines that regulate naïve CD4 T cell activation and differentiation into effector subsets. For example, in response to TLR ligation, DC express the cytokine IL-6, a pro-inflammatory cytokine that is critical for T cell activation. Moreover, DC are known to secrete IL-12 in response to certain TLR4 ligands, such as LPS. IL-12 is pro-T_(H)1 cytokine, and culture of naïve CD4 TA cells with IL-12 and APC leads to their differentiation in vitro into T_(H)1 effector cells. [See, Manetti R, et al. (1993) J Exp Med; April 1; 177(4):1199-204.] DC are also capable of secreting other cytokines, including TGF-β, IL-10, and IL-23, in response to specific stimuli.

In certain embodiments of the invention, the mRNA expression of cytokines and other genes is quantified. For the isolation of RNA, any suitable means known in the art may be used. This includes phenol-chloroform extraction followed by ethanol or 2-propanol precipitation. Certain commercial reagents, such as TRIzol® reagent are well known in the art and may be used according to the manufacturer's instructions for the isolation of RNA.

The present invention, in certain aspects, provides methods for inducing DCs to secrete specific combinations of cytokines, either in vitro or in vivo, based on the discovery of certain stimuli that are capable of inducing such combinations. These stimuli include TLR agonists in combination with apoptotic cells or apoptotic-cell associated agents.

“TLR agonist” or “TLR adjuvant” is understood to mean a natural TLR ligand, a TLR ligand mimic, a synthetic or chemical TLR ligand, a cell or particle including a pathogen-associated molecular pattern, a microbial pathogen, a bacterium, and a virus or viral and viral-like particle. Moreover, it is well known in the art that TLRs may be expressed on the cell surface, or intracellularly, such as, e.g., in endosomes. Moreover, TLRs may function as heterodimers (e.g., as a TLR1/2 and TLR2/6 heterodimers) or as homodimers (e.g., as a TLR4/4 homodimer). TLRs may also function as monomers.

TLR agonists may be synthetic, chemical or nature ligands of TLRs. TLR agonists include, but are not limited to, Pam3CysSerLys4(CSK4) (for TLR1/2); Lipoarabinomannan, LPS P.gingivalis as well as LPS from Gram-positive bacteria, peptidoglycan (PGN) (e.g., from S.aureus), beat killed forms of microbial pathogens such as Listeria monocytogenes (HKLM), lipteichoic acid (LTA) (e.g., from S.aureus), triacylated bacterial lipopeptides (for TLR1/2), diacylated lipopeptides (for TLR2/6), and Malp-2 (for TLR2); Poly(I:C) and dsRNA (for TLR3); LPS (from Gram-negative bacteria such as E. coli) Monophosphoryl lipid A (MPLAp), heat shock protein (HSP) 60, and extra domain A of fibronectin (EDA) (for TLR4); Flagellin (for TLR5); FSL-1 (for TLR2/6); Imiquimod, Loxoribine, Gardiquimod™, and E.coli RNA/LyoVec (for TLR7); ssRNA, polyU, PolyU/LyoVec, Gardiquimod™, and E.coli RNA/LyoVec (for TLR8); CpG oligodeoxynucleotides (ODN) (for TLR9); and profilin from T. gandii and uropathogenic E. coli (for TLR11).

In certain embodiments, the invention relates to compositions for inducing T_(H)17 responses which include an apoptotic cell or apoptotic cell-associated agent. Apoptosis is an active process of cell suicide involving the action of a number of caspase proteins that leads to ordered destruction of the cells and their safe disposal by professional (macrophages and immature DC) and in some cases nonprofessional (such as fibroblasts and epithelial cells) phagocytes. The removal of apoptotic cells in the final step and perhaps the ultimate objective of the apoptotic programs. Apoptosis is a ubiquitous process and plays a key role in many fundamental biological events, including embryonic development, normal tissue homeostasis, development of the immune system and resolution of inflammation. In addition, apoptotic cells are a potential source of self-antigens, and defective clearance of cell corpses has recently been implicated in the pathogenesis of autoimmune disease [Botto et al. (2004) Arthritis Res Ther; 6:147-150].

Dying, apoptotic cells can provide specific signals that enable recruitment and recognition by phagocytes (e.g., DC). Apoptosis is characterized by a variety of morphological feathers such as loss of membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. One of the earliest indications of apoptosis is the translocation of the membrane phospholipid phosphatidylserine from the inner to the outer leaflet of the plasma membrane. Phosphatidylserine is a key apoptosis mediator. Receptors for phosphatidyl serine continue to be discovered, however, they include the phosphatidylserine receptor as well as Tim proteins, such as Tim1 and Tim4. [Miyanishi M, et al (2007) Nature. 450:435-9; Kobayashi N., et al. (2007) Immunity. 27:927-40]. Thus, the present invention contemplates targeting any receptor shown to be involved in the recognition of phosphatidylserine. Once exposed to the extracellular environment, binding sites on phosphatidylserine become available and can recruit phagocytic cells.

In the present invention, the term “apoptotic cell” is understood to include a cell having lost integrity of its plasma membrane, a cell having exposed phosphatidylserine residues, translocated calreticulin to its cell surface, absorbed soluble proteins such as C1q and thrombospondin, or exhibiting other “eat me” signals (i.e., signals to a phagocytic cell to “eat” or phagocytose the apoptotic cell), a cell having suppressed “do not eat me” (i.e., “do no phagocytose me”) signals such as suppression of surface CD47 expression, a cell having complete permeabilization of its outer mitochondrial membrane, a cell having lost mitochondrial membrane potential, a cell having caspase activation, a cell having ΔΨm dissipation, a cell having a nucleus that has undergone complete fragmentation into discrete bodies frequently referred to as “apoptotic bodies” and measured by quantification of hypodiploid events (sub-G1 peak), a cell having nuclear fragmentation (karyorrhexis), a cell labeling positively for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), a cell triggered to undergo apoptosis through different biochemical routes (the “intrinsic” or the “extrinsic” pathway, with or without the contributions of mitochondria), a cell exhibiting typical apoptotic morphology define by rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), chromatin condensation, karyorrhexis, little or no ultrasound modifications of cytoplasmic organelles, plasma membrane blebbing (but maintenance of its integrity until the final stages of the process) and engulfment by phagocytes.

As used herein, the term “apoptotic cell-associated agent” refers to an entire apoptotic cell, or, to molecular components of an apoptotic cell, no phospholipids (e.g., phosphatidylserine) and intracellular proteins such as calreticulin or polypeptides that are exposed outside their normal intracellular compartments during apoptotic, (i.e., on the outer cell membrane), to synthetic apoptotic cell mimics, or to any apoptotic cell derived ligand recognized by a receptor specific to the apoptotic cell derived ligand and expressed by the DC or macrophage. This apoptotic cell-associated agent or mimic would trigger specific receptors triggering the same signaling pathways that an apoptotic cell would otherwise trigger in a DC, macrophage or other phagocytic cell, recognizing that apoptotic cell or engaged in phagocytosis of that apoptotic cell.

In certain embodiments of the invention, cell lines are used to study apoptosis. For example, the B cell lymphoma cell line A20 (ATCC number TIB-208) may be treated with anti-Fas antibody (anti-CD95) to induce apoptosis. The Fas receptor (also known as Apo-1 or CD95) binds the Fas ligand (FasL), a transmembrane protein part of the TNF family. The interaction between Fas and FasL results in the formation of the death-inducing signaling complex (DISC), which contains the FADD, caspase-8 and caspase-10. In some types of cells, processed caspase-8 directly activates other members of the caspase family, and triggers the execution of apoptosis. In other types of cells, the Fas-DISC starts a feedback loop that spirals into increasing release of pro-apoptotic factors from mitochondria and the amplified activation of caspase-8. Thus, cells, such as A20 cells, express Fas ligand and their cell surface undergo apoptotic cell death upon cross-linking of Fas, either by Fas ligand or by anti-Fas antibodies.

In a highly preferred embodiment of the present invention, the TLR ligand is incorporated directly into the apoptotic cell. The TLR ligand may be recognized by cell surface TLR, such as e.g., TLR-1, 2, or 4, or may be recognized by an endosomal TLR, such as, e.g. TLR-3, 7, or 8.

In other embodiments, apoptotic neutrophils are generated in vivo by the intraperitoneal injection of thiogylcollate. Upon thiogylcollate injection, neutrophils are recruited to the inflamed injection site. Once, recruited, neutrophils which are short-lived cells, live only a matter of days, and soon begin undergoing apoptosis. These cells may be isolated and used for study. Thiogylcollate may also be mixed with live E. coli and this mixture can be injected intraperitoneally. In this case, neutrophils recruited to the peritoneal cavity will phagocytose the injected E. coli and under apoptosis. These latter neutrophils differ from the former (without E. coli) in that they have served in the studies supporting the present invention as a model for an infected apoptotic cell since they have phagocytosed E. coli and now undergo apoptosis carrying both signatures of TLR ligands and apoptotic cells.

In certain aspects of the invention, cell apoptosis is detected. Apoptosis may be detected by a variety of methods known in the art, including, but not limited to flow cytometric-based analysis of Annexin V expression on the cell surface. Annexin V is a 35-36 kDa. Ca²⁺-dependent, phospholipid binding protein with a high affinity for phosphatidylserine. The translocation of phosphatidylserine precedes other apoptotic processes such as loss of plasma membrane integrity, DNA fragmentation, and chromatin condensation. As such, Annexin V can be conjugated to biotin or to fluorochrome such as FITC, PE, APC, Cy5, or Cy5.5, and used for the easy, flow cytometric indentification of cells in the early stages of apoptosis.

Because phosphatidylserine translocation also occurs during necrosis, Annexin V is not an absolute marker of apoptosis. Therefore, it may be used in conjunction with vital dyes such as 7-amino-actinomycin D (7-AAD) or propidium iodide (PI), which bind to nucleic acids, but only penetrate the plasma membrane when membrane integrity is breached, as occurs in the later stages of apoptosis or in necrosis. Using these methods, cells that are negative for both Annexin V and the vital dye have no indications of apoptosis, since phosphatidylserine translocation has not occurred and the plasma membrane is still intact. Cells that are Annexin V-positive and vital dye-negative, however, are in early apoptosis as phosphatidylserine translocation has occurred, yet the plasma membrane is still intact. Cells that are positive for both Annexin V and the vital dye are either in the late stages of apoptosis or are already dead, as phosphatidylserine translocation has occurred and the loss of plasma membrane integrity is observed. When measured over time, Annexin V and a vital dye can be used to monitor the progression of apoptosis from cell viability, to early-stage apoptosis, and finally to late-stage apoptosis and cell death.

In certain other embodiments of the invention, apoptosis may also be assessed using TUNEL staining. The TUNEL (terminal deoxynucleotide transferase [TdT]-mediated dUTP-digoxigenin nick-end labeling) method is based on the in situ labeling of DNA fragmentation sites in nuclei of intact fixed cells. DNA fragmentation is characteristic of apoptotic cells. TUNEL staining may be performed using the In Situ Cell Death Detection Kit, TMR Red (Roche, Indianapolis, Ind.).

The use of synthetic mimics for the effects of apoptotic cells may be in the form of phosphatidylserine incorporated into liposomes, or agonists for nuclear hormone receptor superfamily member. Peroxisome Proliferators-Activated Receptor (PPAR)-γ. A combination of TLR ligands with PPAR-γ agonists, for example, thiazolidinediones (also referred to as glitazones) such as Rosiglitazone, are examples of such synthetic agonists. Thiazolidinediones are used as therapy against various inflammatory diseases because of their anti-inflammatory and anti-proliferative effects. They are also used for treatment of type 2 diabetes. Moreover, it has been reported that recognition of apoptotic cells by cells of the innate immune system, such as macrophages, results in the activation of PPAR-γ [Johann A. M. et. al (2006) Cell Death Diff; 13:1533-1540].

In certain other embodiments, a mimic of cell surface calreticulin translocation, including but not limited to inhibitors of the protein phosphatase 1/GADD34 complex, may be used to mimic cellular apoptosis.

In certain embodiments of the invention, an apoptotic cell or a microbe-infected apoptotic cell is engineered to express an exogenous immune antigen. As used herein “exogenous” refers to a factor that is present and active in an individual organism or living cell but that originated outside of that organism, as opposed to an “endogenous” factor, which originates from the organism expressing the factor. As used herein, an “exogenous immune antigen” expressed by a microbe or expressed by a microbe-infected apoptotic cell refers to an antigen that is not endogenously expressed by the apoptotic cell. In certain embodiments, it is preferred that the exogenous immune antigen is expressed neither by the apoptotic cell nor by the microbe infecting the appropriate cell. In other embodiments, it is preferred that the exogenous immune antigen is expressed endogenously by the microbe but is not expressed endogenously by the apoptotic cell, DNA introduced to cells via transfection or viral infection (transduction) is a non-limiting example of an exogenous factor. This method can be used to express as exogenous antigen by an apoptotic cell which is otherwise not expressed by the apoptotic cell. Non-limiting examples of an exogenous immune antigen include a tumor-associated antigen, and an antigen expressed by a microbe that could be used to elicit a T_(H)17 immune response to the microbe, e.g., to induce effective host defense against the microbe as well as tissue repair. In certain embodiments of the invention, such as, e.g., vaccine compositions of the invention, an “immune antigen” is provided. As used herein, an “immune antigen” may be any antigen that elicits an immune response, and may be either an exogenously or endogenously express antigen. Endogenous or exogenous (e.g. tumor associated antigen) expressed antigens would be used in the context of apoptotic cells alone as a regulatory T cell adjuvant where a regulatory T cell response is preferentially induced, while an exogenously expressed antigen such as a microbial antigen or a tumor-associated antigen would be used in the context of an apoptotic cell engineered to carry TLR ligands as a T_(H)17 adjuvant where a T_(H)17 immune response is preferentially induced.

Apoptotic cells may be engineered to express such exogenous immune antigens by any suitable method known in the art. Examples of such methods include transfecting a suitable cell line or virus with the gene encoding the desired antigen or transforming a cell, such as, e.g., a bacterium, with the gene encoding the desired antigen using a suitable virus, e.g. a lentivirus or an adenovirus. Apoptosis may then be induced in the transfected or transformed cell before use. In other embodiments, a certain apoptotic cell may be selected for use because it endogenously (i.e., naturally) expresses a desired immune antigen. In other embodiments, a cell expressing a desired immune antigen is either infected with a microbe, such as, e.g., E. coli, or allowed to internalize an inactivated form of a microbe such as E. coli. Apoptosis is then induced following infection or internalization of the microbe. In other embodiments, a cell can be made to express an antigen as a fusion protein with a TLR ligand such as Flagellin. Apoptosis is then induced, where the resultant apoptotic cell expresses the exogenous antigen directly fused to a TLR ligand. Apoptosis may be induced by a number of methods known in the art, including exposure to heat, UV, or fixation. The specific method of apoptosis induction will depend on the type of cell to be treated, and is readily determined by a skilled artisan.

According to certain embodiments of the present invention, the combinatorial use of PPAR-γ agonists and TLR ligands provides a stimulus for T_(H)17 cell development. Specifically, PPAR-γ agonists and TLR ligands may be administered as one entity such as encapsulated within liposomes, e.g., in order to achieve co-internalization by DC into one subcellular compartment such as an endosome or phagosome.

In certain embodiments of the present invention, apoptosis may be inhibited using Q-VD-OPH, a broad-spectrum caspase inhibitor. Q-VD-OPH is not toxic to cell even at extremely high concentrations, and consists of a carboxy terminal phenoxy group conjugated to the amino acids valine and aspartate. The compound is a potent inhibitor of cell death by apoptosis and is significantly more effective in preventing apoptosis than ZVAD-fmk and Boc-D-fmk. It is equally effective in preventing apoptosis mediated by the three major apoptotic pathways, caspase-9 and caspase-3, caspase-8 and caspase-10, and caspase-12. [Caserta T M et al (2003) Apoptosis 8(4): 345-352; Patil K. and Sharma S C (2004) NeuroReport 15:981-984; Yang L et al. (2004) Neurobiology of Disease 17(2): 250-259].

In some aspects, the present invention relates to compositions, pharmaceutical formulations or vaccine compositions that include specific combinations of the TLR agonists and apoptotic cells or apoptotic cell-associated agents described herein. In certain embodiments, TLR agonists and apoptotic cells are combined as a single entity by coadministration in one physical form as described herein. For example, a TLR ligand and an apoptotic cell-associated agent may be incorporated into liposomes such that each liposome carries both an apoptotic cell-associated agent (e.g., phosphatidylserine) and a TLR ligand. This composition endures co-delivery in one physical form and internalization into one subcellular compartment within DC (e.g., endosome or phagosome). In other embodiments, the combination of the two T_(H)17 adjuvants occurs naturally, and is administered as a single agent, such as, for example, a microbe-infected apoptotic cell or an apoptotic cell engineered to carry a TLR ligand. A microbe-infected apoptotic cell according to the present invention expresses markers of apoptosis suitable for eliciting TGF-β secretion by DC and TLR ligands suitable for eliciting IL-6 secretion by the same DC.

Any form or method of delivery, either in vitro or in vivo, that results in co-internalization of both the TLR agonist and the apoptotic cell-associated agent by the same DC, or that results in stimulation of a DC such that the DC is induced to become a T_(H)17-inducing DC by exposure to the TLR agonist and the apoptotic cell-associated agent, is contemplated by the present invention.

Non-limiting examples of microbe-infected cells that may be used in the present invention include cells infected with live attenuated vaccine strains such as Mycobacterium bovis BCG vaccine against tuberculosis, Salmonella typhi Ty21a vaccine against typhoid fever, and Vibrio cholerae CVD 103-HgR vaccine against cholera. Killed bacteria of all strains may also be used after inactivation with heat, UV or fixation. These may then be given to phagocytic cells and apoptosis of the cell induced after internalization of the inactivated microbe. In certain embodiments of the present invention, neutrophils infected with E. coli are used. E coli infection of neutrophils causes them to become apoptotic, and the E. coli provides TLR ligands, thus representing an agent which combines both of the two necessary T_(H)17 adjuvants.

Bacteria that cause significant apoptosis and tissue damage at epithelial surfaces during infection include, but are not limited to, Shigella dysenteriae, Klebsiella pneumoniae, Pseudomonas aeruginosa, enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC)

In certain embodiments, the invention relates to compositions that have a DC capable of inducing a T_(H)17 response, wherein the DC secretes IL-6 and TGF-β. The DC compositions may also include a microbe-infected apoptotic cell. The DC may phagocytose the microbe-infected apoptotic cell expressing exogenous immune antigens also administered to the DC. A killed microbe (inactivated, e.g., by heat, UV or fixative) may also be used because the DC will phagocytose this microbe, a method which delivers to the DC not only the relevant adjuvants for T_(H)17 induction but also all the exogenous immune antigens unique to that microbe. Other microbes may include those selected for use as live attenuated vaccine strains such as Mycobacterium bovis BCG vaccine against tuberculosis, Salmonella typhi Ty21a vaccine against typhoid fever, and Vibrio cholerae CVD 103-HgR vaccine against cholera.

In certain embodiments, DC are generated in vitro in bone marrow (BM)-derived GM-CSF DC cultures. Using this method, large numbers of BMDC may be generated by culturing whole BM cells in the presence of GM-CSF. The cultures are grown in RPMI supplemented with GM-CSF and 5% foetal bovine serum (FBS), plus 100 μg/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 nM sodium pyruvate, 1X MEM nonessential amino acids, and 2.5 μM β-mercaptoethanol (all from Sigma-Aldrich, St. Louis, Mo.). Semi-adherent cells are then harvested on ice on day 5 and re-plated immediately in fresh GM-CSF medium at 1×10⁶ cells/well in 24-well tissue culture-treated plates and used for experiments.

In the present invention, other phagocytic cells in addition to DC that are capable of inducing T_(H)17 responses are also contemplated.

In certain embodiments of the present invention, in vitro experiments are carried out to assess the T_(H)17-inducing potential of a DC. In certain embodiments, a DC is co-cultured directly with a T cell in vitro in the presence of a TLR agonist and apoptotic cell-associated stimuli of the present invention. In other embodiments, the DC is first treated with a TLR adjuvant and apoptotic cell-associated stimuli of the present invention, and then the DC conditioned media (CM) is collected and transferred to a well containing CD4 T cells stimulated with T cell activating agents (e.g., anti-CD3 and anti-CD28 antibodies). The ability of a soluble factor secreted by the DC into the conditioned media to induce CD4 T cell differentiation may then be assessed. Such in vitro methods are widely accepted in the art as suitable models for characterizing the requirements for CD4 T cell differentiation in vivo.

In certain embodiments of the present invention, T cell proliferation may be determined by measuring tritiated thymidine (³H-Thymidine) incorporation into dividing cells. As T cells proliferate, they incorporate the labeled nucleic acid into the dividing cells. The resulting radioactivity of the divided cells can be detected using a beta liquid scintillation counter. In the examples of the present disclosure, following 72 hours of co-culture, 1 μCi of ³H-thymidine is added to each culture well. Then, 18 hours after pulsing with ³H-thymidine, cells are harvested with a multiple-sample harvester and counted with a Wallac 1450 microbeta PLUS liquid scintillation counter (Perkin-Elmer, Waltham, Mass.). Using this approach, the ability of DC to stimulate T cell proliferation can be quantitated.

In certain embodiments of the present invention, naïve CD4 T cells are isolated by cell sorting. Naïve CD4 T cells are identified by the levels of expression of certain cell surface markers. Specifically, these cells are CD62L^(high) CD44^(low) CD25⁻ cells that can be isolated by fluorescence activated cell sporting (FACS). The CD4 T cells are strained with fluorescently-conjugated antibodies specific for epitopes of CD62L, CD44, and CD25, and sorted based on their fluorescence properties to isolate the CD62L^(high) CD44^(low) CD25⁻ population. This approach yields highly pure (>99%) populations of naïve CD4 T cells.

In certain embodiments, intestinal cell damage is induced using dextran sulfate sodium (DSS). DSS induces an inflammatory bowel disease-like colitis in animals. In addition to using DSS for the study of inflammation, numerous animal models exist for the study of colorectal and intestinal cancers in which mice are genetically manipulated or challenged with chemicals, such as DSS, to develop malignancies in the gastrointestinal tract. These models enable researchers to study the onset, progression of the disease, and understand in depth the molecular events that contribute to the development and spread of colorectal cancer. For example, human Inflammatory Bowel Disease (IBD) is a group of inflammatory conditions in the large and small intestine. It is well known that chronic inflammation in the colon can cause cancer. Genetic mouse models for IBD-associated colon cancer include a model in which IL-10 knock out mice develop invasive adenocarcinoma in the colon, a model in which mice that are mutant for IL-2 and beta microglobulin genes have ulcerative colitis-like phenotypes and develop adenocarcinomas in the colon, and a model in which a mouse mutant for N-cadherin suffers IBD conditions and adenomas but does not develop carcinomas.

Compositions and Methods for Inhibiting a T_(H)17 Response or Inducing Immune Tolerance

In certain embodiments, the present invention relates to methods for inhibiting T_(H)17 responses, and in other embodiments, the invention relates to methods for inducing immune tolerance in a mammal. These methods can involve administering to the DC an apoptotic cell in the absence of TLR ligands (e.g., an uninfected apoptotic cell), or administering an agent that blocks immune recognition of an apoptotic cell, or an apoptotic cell-associated agent. In yet other embodiments, an agent that blocks immune recognition of a Toll-like receptor (TLR) ligand or adjuvant may be administered. The present invention also relates to compositions and pharmaceutical formulations including one ore more blocking reagents useful for inhibiting T_(H)17 responses and/or for inducing immune tolerance (i.e. Treg responses).

Examples of suitable blocking agents include, but are not limited to antibodies that block recognition of apoptotic cells, or agents associated with apoptotic cells, such as, e.g., phosphatidylserine, an antibodies that block recognition of TLR ligands. Specific inhibitors of TLR signaling include but are not limited to novel small molecule TLR4 antagonists such as TAK-242 [M. Ii, N. et al (2006) Mol Pharmacol 69: 1288], Eritoran (E-5564), an antagonistic version of the lipid A portion of LPS that binds TLR4 and acts as an antagonist (ref2), small molecule inhibitors of IKK2 (ref 3), NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) [M. Säemann et al (2004) Am J Transplant 4:1448], NF-κB oligodeoxyribonucleotide decoys or RNA for silencing the NF-κB genes [C. A. Bonham et al. (2002) J Immunol 169:3382; M. Li et. al., (2006) Am J Transplant (suppl), p. 311 WTC-Congress Boston, abstract #725], agents that block endosomal acidifications, such as chloroquine or hydroxychloroquine thereby blocking signaling by intracellular TLRs, vaccine virus derived proteins A46R and A52R, anti-inflammatory agents such as aspirin, salicylate and other non-steroidal anti-inflammatory drugs (NSAIDS), glucocorticoids which interfere with NF-κB-mediated gene transcription, natural products such as parthenolide that block IKK2, NEMO-binding peptides, PS-1145 a proteoasome inhibitor that blocks IκB degradation, IRAK-4 inhibitors, inhibitors of protein-protein interactions that might be used to inhibit TIR domain interactions (e.g., MyD88 recruitment to TLRs or IRAK-1/TRAF-6 interactions), and inhibition of TRAF-6 and TAK-1 ubiquitination. [reviewed in O'Neill LA. (2003) Curr Opin Pharmacol; 3:396-403].

Agents that block recognition of apoptotic cells include but are not limited to monoclonal antibodies (mAb) that serve to inhibit phosphatidylserine-dependent phagocytosis of apoptotic cells in vitro. one mAb, Kat 5-19, which is specific to the newly identified phosphatidylserine receptor, the T cell immunoglobulin mucin-4 (Tim-4), can do so in vitro and in vivo by specifically blocking spontaneous ingestion of apoptotic cells by macrophages [M. Miyanishi, et al (2007) Nature 450; 435-439]. Other neutralizing antibodies that block recognition of phsophatidylserine have also been described [Kobayashi N, et. al (2007) Immunity, 27: 927-940]. Recognition of phosphatidylserine is likely complex and possibly involves both thrombospondin (TSP) and GAS6 which might bridge apoptotic cell phosphatidylserine to phagocyte αv integrins and Mer kinase, respectively. Blocking the ability of these molecules to form a bridge between phosphatidylserine and the DC may be an effective means of blocking apoptotic cell recognition. Finally, milk fat globule epidermal growth factor 8 (MFG-E8) has also been identified as a bridging molecule between apoptotic cell phosphatidylserine and the DC αyintegrins, an interaction which may also be blocked to prevent recognition of apoptotic cell exposed phosphatidylserine [Reviewed in Savill and Gregory (2007) Immunity 2007 27:830-832].

In some aspects of the present invention, methods for the treatment of a T_(H)17-driven disease or condition are provided. Diseases or conditions that may benefit from the compositions and methods of the present invention include, but are not limited to, inflammatory bowel disease, Crohn's disease, colitis, systemic sclerosis (scleroderma), atopic dermatitis, psoriasis, rheumatoid arthritis, diabetes, cystic fibrosis, allergic airway disease, atopic asthma, allergic asthma, Sjogren's Syndrome, and systemic lupus erythematosus.

Vaccine Compositions

The vaccine compositions of the present invention may be used to induce T_(H)17 responses in a patient. Alternatively, the vaccine compositions of the present invention may be used to inhibit T_(H)17 responses and, optionally, to induce T_(reg) cell responses.

In certain embodiments, the vaccine compositions of the present invention preferentially induce T_(H)17 responses based on the novel combination of adjuvants which they contain. Specifically, in certain embodiments, the vaccine compositions contain a TLR ligand-containing apoptotic cell or an apoptotic cell-associated agent preferably administered with a TLR ligand as a single entity or physical form. Conventionally, vaccine compositions contain adjuvants, such as cholera toxin, that stimulate the immune response. Many of the adjuvants that are safe for human use do not elicit effective immune responses, in part because they do not specifically elicit T_(H)17-driven responses in mucosal sites. Most vaccines are delivered to mucosal sites, either via the airways or orally, where T_(H)17 responses are thought to be preferred. Thus, the present invention provides vaccine compositions which preferentially induce T_(H)17 responses.

For the treatment of certain diseases, preferential induction of T_(H)17 responses by a vaccine composition is highly preferred. For example, as discussed, supra, certain cancers, such as epithelial or mixed epithelial carcinomas, are adversely effected by the presence of T_(reg) cells, which may prevent successful immune responses against the tumor. In this case, the compositions and methods provided by the present invention, which preferentially induce a T_(H)17 response, thereby inhibiting the T_(reg) response, are highly useful for the treatment of certain cancers. Similarly, infections with certain strains of bacteria that cause significant apoptosis and cell death in infected tissues may preferentially benefit from the induction of T_(H)17 responses. These may include but are not limited to infections with Pseudomona aeruginosa, Klebsiella pneumoniae, Shigella dysenteriae, and enteropathogenic or enterohemorrhagic E. coli (EPEC and EHEC, respectively).

In certain other embodiments, the vaccine compositions of the present invention inhibit T_(H)17 responses. This is achieved by the selective inhibition of T_(H)17 response by blocking recognition of either one or both of the TLR ligand and the apoptotic cell.

In certain other embodiments, the vaccine compositions of the present invention inhibits T_(H)17 responses and instead induced T_(reg) responses. This is achieved by the selective inhibition of the T_(H)17 response by blocking the TLR stimulating component of the TLR ligand-containing apoptotic cell adjuvant discovered by the present invention. This results in activity of the apoptotic cell alone (without TLR ligand), a scenario that favors T_(reg) responses and thus serves to modulate T_(H)17 responses to T_(reg) responses.

Vaccine compositions which inhibit T_(H)17 responses are highly useful for the treatment of autoimmune diseases, such as inflammatory bowel disease, Crohn's disease, colitis, systemic sclerosis (scleroderma), atopic dermatitis, psoriasis, rheumatoid arthritis, diabetes, cystic fibrosis, allergic airway disease, atopic asthma, allergic asthma, Sjogren's Syndrome, and systemic lupus erythematosus. Such vaccine compositions provided by the present invention are also highly useful for the treatment of certain cancers, in which it is useful to inhibit T_(H)17 responses, and to increase the number of T_(reg) cells. As discussed, supra, such cancers include, but are not limited to, Hodgkin lymphoma, follicular lymphoma, multiple myeloma, monoclonal gammopathy, and T cell leukemia/lymphoma,

In certain embodiments of the invention, compositions and formulations of the invention, including vaccine compositions, contain DC along with the combination of a TLR adjuvant and an apoptotic cell component preferably administered as a single entity or in one physical form. The present invention discloses the surprising finding that loading DC, either during or before transfer to a patient, with the combination of a TLR agonist and an apoptotic cell or apoptotic cell-associated agent (as one entity, e.g., encapsulated in liposomes or physically linked or contained within a carrier), can generate a T17 response in vivo. “Loading” of a DC means that the DC is cultured en vivo or in vitro and pulsed with the cargo with which the DCs is to be “loaded”. For example, in certain embodiments, the DC is pulsed with a microbe-infected apoptotic cell or an apoptotic cell that previously internalized as inactivated form of a microbe. Upon phagocytosis of the infected apoptotic cell, the DC concurrently recognizes the TLR ligands associated with the microbe infecting the cell and that the cell is apoptotic (e.g., through receptor-mediated binding of phosphatidylserine), and thus becomes primed to induce a T_(H)17 response upon transfer to the recipient (patient). Moreover, if the DC is also loaded with a suitable vaccine antigen, e.g., a tumor-associated antigen, the DC will present peptides of this antigen in the context of surface MHC class II molecules to CD4 T cells in the patient, thereby inducing an antigen-specific, T_(H)17-driven immune response.

Definitions

The following definitions are provided for the clarity of illustrative purposes only, and are not intended to limit the scope of the invention.

Expression Construct

By “expression construct” is meant a nucleic acid sequence comprising a target nucleic acid sequence or sequences whose expression is desired, operatively associated with expression control sequence elements which provide for the proper transcription and translation of the target nucleic acid sequence(s) within the chosen host cells. Such sequence elements may include a promoter and a polyadenylation signal. The “expression construct” may further comprise “vector sequences”. By “vector sequences” is meant any of several nucleic acid sequences established in the art which have utility in the recombinant DNA technologies of the invention to facilitate the cloning and propagation of the expression constructs including (but not limited to) plasmids, cosmids, phage vectors, viral vectors, and yeast artificial chromosomes.

Expression constructs of the present invention may comprise vector sequences that facilitate the cloning and propagation of the expression constructs. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic host cells. Standard vectors useful in the current invention are well known in the art and include (but are not limited to) plasmids, cosmids, phage vectors, viral vectors, and yeast artificial chromosomes. The vector sequences may contain a replication origin for propagation in Escherichia coli (E. coli); the SV40 origin of replication; an ampicillin, neomycin, or puromycin resistance gene for selection in host cells; and/or genes (e.g., dihydrofolate reductase gene) that amplify the dominant selectable marker plus the gene of interest.

Express and Expression

The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g., the resulting protein, may also be said to be “expressed” by the cell. An expression product can be characterized as intracellular, extracellular or secreted. The term “intracellular” means something that is inside a cell. The term “extracellular” means something that is outside a cell. A substance is “secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.

Transfection

The term “transfection” means the introduction of a foreign nucleic acid into a cell. The term “transformation” means the introduction of a “foreign” (i.e. extrinsic or extracellular) gene. DNA or RNA sequence to a cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by cells genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been “transformed” and is a “transformant” or a “clone”. The DNA or RNA introduced to a host cell can come from any source, including cells of the same genes or species as the host cell, or cells of a different genus or species. In certain embodiments of the present invention, for example, MFB-F11 mouse fibroblast cells are stably transfected with a reporter plasmid consisting of TGF-β-responsive Smad-binding elements coupled to a secreted alkaline phosphatase reporter gene (SBE-SEAP).

Electroporation

“Electroporation”, as used herein, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.

Expression System

The term “expression system” means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

Gene or Structural Gene

The term “gene”, also called a “structural gene” means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulator of DNA transcription.

A coding sequence is “under the control of” or “operatively associated with” expression control sequences in a cell when RNA polymerase transcribes the coding sequence into RNA, particularly mRNA, which is then trans-RNA spliced (if it contains introns) and translated into the protein encoded by the coding sequence.

The term “expression control sequence” refers to a promoter and any enhancer or suppression elements that combine to regulate the transcription of a coding sequence. In a preferred embodiment, the element is an origin of replication.

Protein or Polypeptide

The definitions of protein and polypeptide are well-known in the art. The term “protein”, as used herein, is synonymous with the term “polypeptide”, and is understood to mean a chain of amino acids arranged linearly and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.

Heterologous

The term “heterologus” refers to a combination of elements not naturally occurring. For example, heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA includes a gene foreign to the cell. For example, the present invention includes chimeric DNA molecules that comprise a DNA sequence and a heterologous DNA sequence which is not part of the DNA sequence. A heterologous expression regulatory element is such an element that is operatively associated with a different gene than the one it is operatively associated with in nature. In the context of the present invention, a gene encoding a protein of interest is heterologous to the vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed.

Homologous

The term “homologous” as used in the art commonly refers to the relationship between nucleic acid molecules or proteins that possess a “common evolutionary origin,” including nucleic acid molecules or proteins within superfamilies (e.g., the immunoglobulin superfamily) and nucleic acid molecules or proteins have sequence (Reeck et al., Cell 1987; 50: 667). Such nucleic acid molecules or proteins have sequence homology, as reflected by their sequence similarity, whether in terms of substantial percent similarity or the presence of specific residues or motifs at conserved positions.

Host Cell

The term “host cell” means any cell of any organism that is selected, modified, transformed, grown or used or manipulated in any way for the production of a substance by the cell. For example, a host cell may be one that is manipulated to express a particular gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays that are described infra. Host cells may be cultured in vitro or one or more cells in a non-human animal (e.g., a transgenic animal or a transiently transfected animal). Suitable host cells include but are not limited to Streptomyces species and E. coli.

Microbe or Pathogen

The terms “microbe” and “microorganism” are understood to include, but are not limited to, bacteria, viruses, fungi, archaea, and protists. One of ordinary skill in the art will understand that the term “microbe” applies to any biological microscopic organism. A pathogen is typically defined as an organism or microbe, such as a bacterium or virus that can invade a host and cause harm to the host. Intracellular and extracellular parasites may also be pathogens. Usually, a pathogen will elicit an immune response in the infected host. Some microbes are not normally pathogenic, such as, e.g., the bacteria that line the gut or the surface of the epithelium (skin), however, even conventionally non-pathogenic bacteria may become pathogenic in certain circumstances, e.g., if they become overpopulated, or if they colonize sites of the body that they normally do not populate. The present invention identifies that bacteria which cause significant apoptosis and tissue damage at epithelial surfaces are most likely best suited to induce T_(H)17 immunity. Apoptosis is typically caused by special type III secretion systems that inject bacterial apoptosis effector proteins into the host cell.

Treating or Treatment

(1) Preventing or delaying the appearance of clinical or sub-clinical symptoms of the state, disorder or condition developing in a mammal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or

(2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at lease one clinical sub-clinical symptom thereof; or

(3) Relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms.

The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

Patient or Subject

“Patient” or “subject” refers to mammals and includes human and veterinary subjects.

Therapeutically Effective Amount

A “therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a state, disorder or condition, is sufficient to effect such treatment. The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, physical condition and responsiveness of the mammal to be treated.

Prophylactically Effective Amount

A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic does is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

About or Approximately

The term “about” or “approximately” means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Unless otherwise stated, the term ‘about’ means within an acceptable error range for the particular value.

Include or Comprise

As used herein, the terms “include” and “comprise” are used synonymously. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

Isolated

As used herein, the term “isolated” means that the referenced material is removed from the environment in which it is normally found. Thus, an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced. Isolated nucleic acid molecules include, for example, a PCR product, an isolated mRNA, a cDNA, or a restriction fragment. Isolated nucleic acid molecules also include, for example, sequences inserted into plasmids, cosmids, artificial chromosomes, and the like. An isolated nucleic acid molecule is preferably excised from the genome in which it may be found, and more preferably is no longer joined to non-regulatory sequences, non-coding sequences, or to other genes located upstream or downstream of the nucleic acid molecule when found within the genome. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.

Purified

The term “purified” as used herein refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e. contaminants, including native materials from which the material is obtained. The isolated material is preferably substantially free of cell or culture components, including tissue culture components, contaminants, and the like. As used herein, the term “substantially free” is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure, more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.

Mutant

As used herein, the terms “mutant” and “mutation” refer to any detectable change in genetic material (e.g., DNA) or any process, mechanism, or result of such a change. This includes gene mutations, in which the structure (e.g., DNA sequence) of a gene is altered, any gene or DNA arising from any mutation process, and any expression product (e.g., protein or enzyme) expressed by a modified gene or DNA sequence. As used herein, the term “mutating” refers to a process of creating a mutant or mutation.

Nucleic Acid Molecule

A “nucleic acid molecule” or “oligonucleotide” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine, “DNA molecules”), or any phophoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear (e.g., restriction fragments) or circular DNA molecules, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.

The nucleic acid molecules of sequences disclosed herein are written according to The International Union of Pure and Applied Chemistry (IUPAC) DNA codes. Specifically, “A” is Adenine, “C” is Cystosine, “G” is Guanine, “T” is Thymine, “U” is Uracil, “R” is any Purine (A or G), “Y” is any Pyrimidine (C, T, or U), “M” is C or A, “K” is T, U, or G, “W” is T, U, or A, “S” is C or G, “B” is C, T, U or G (not A), “D” is A, T, U, or G (not C), “H” is A, T, U, or C (not G), “V” is A, C, or G (not T, not U), and “N” is any base (A, C, G, T, or U).

Nucleic Acid Hybridization

The term “nucleic acid hybridization” refers to anti-parallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G. Nucleic acid molecules are “hybridizable” to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under “low stringency” conditions, a greater perecentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid.) See Molecular Biology of the Cell, Alberts et al., 3rd ed., New York and London: Garland Publ., 1994, Ch. 7.

Typically, hybridization of two strands at high stringency requires that the sequences exhibit a high degree of complementarity over an extended portion of their length. Examples of high stringency conditions include: hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% SDS, 1 mM EDTA at 65° C., followed by washing in 0.1x SSC/0.1% SDS at 68° C. (where 1x SSC is 0.15M NaCl, 0.15M Na citrate) or for oligonucleotide molecules washing in 6xSSC/0.5% sodium pyrophosphate at about 37° C. (for 14 nucleotide-long oligos), at about 48° C. (for about 17 nucleotide-long oligos), at about 55° C. (for 20 nucleotide-long oligos), and at about 60° C. (for 23 nucleotide-long oligos)). Accordingly, the term “high stringency hybridization” refers to a combination of solvent and temperature where two strands will pair to form a “hybrid” helix only if their nucleotide sequences are almost perfectly complementary (see Molecular Biology of the Cell, Alberts et al., 3rd ed., New York and London: Garland Publ., 1994, Ch. 7).

Conditions of intermediate or moderate stringency (such as, for example, an aqueous solution of 2XSSC at 65° C., alternatively, for example, hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C.) and low stringency (such as, for example, an aqueous solution of 2XSSC at 55° C.), require correspondingly less overall complementarity for hybridization to occur between two sequences. Specific temperature and salt conditions for any given stringency hybridization reaction depend on the concentration of the target DNA and length and base composition of the probe, and are normally determined empirically in preliminary experiments, which are routine (see Southern, J. Mol. Biol. 1975; 98: 503; Sambrook et al., Molecular Closing: A Laboratory Manual, 2nd ed., vol 2, ch. 9.50, CSH Laboratory Press, 1989; Ausubel et al (eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).

As used herein, the terms “standard hybridization conditions” refers to hybridization conditions that allow hybridization of sequences having at least 75% sequence identity. According to a specific embodiment, hybridization conditions of higher stringency may be used to allow hybridization of only sequences having at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.

Nucleic acid molecules that “hybridize” to any desired nucleic acids of the present invention may be of any length. In one embodiment, such nucleic acid molecules are at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, and at least 70 nucleotides in length. In another embodiment, nucleic acid molecules that hybridize are of about the same length as the particular desired nucleic acid.

Techniques to isolate and modify specific nucleic acids and proteins are well known to those of skill in the art. In accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989 (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription and Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobolized Cells and Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide to Molecular Cloning (1984); Ausubel, F. M. et al (eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include site directed mutagenesis employing oligonucleotides with altered nucleotides for generating PCR products with mutations (e.g., the “Quickchange” kit manufactured by Stratagene).

Primers

The term “primer,” as used herein, refers to an oligonucleotide capable of acting as a point of initiation of DNA synthesis under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., either in the presence of four different nucleoside triphosphates and an agent for extension (e.g., a DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. A primer is preferably a single-stranded DNA. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 6 to 50 nucleotides, preferably from 15-35 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for the amplification of a given target sequence is well known in the art and described in the literature cited herein. As used herein, a “forward primer” is understood to mean a primer that is capable of hybridizing to a region of DNA along the 5′ (coding) strand of DNA. A “reverse” primer is understood to mean a primer that is capable of hybridizing to a region of DNA along the 3′ (non-coding) strand of DNA.

As used herein, a primer is “specific,” for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer hybridizes primarily only the target nucleic acid. Typically, a primer is specific for a target sequence if the primer-target duplex stability is greater than the stability of a duplex formed between the primer and any other sequence found in the sample. One of skill in the art will recognize that various factors, such as salt conditions as well as base composition of the primer and the location of the mismatches, will affect the specificity of the primer, and that routine experimental confirmation of the primer specificity will be need in most cases. Hybridization conditions can be chosen under which the primer can form stable duplexes only with a target sequence. Thus, the use of target-specific primers under suitably stringent amplification conditions enables the specific amplification of those target sequences which contain the target primer binding sites. The use of sequence-specific amplification conditions enables the specific amplification of those target sequences which contain the exactly complementary primer binding sites.

A “primer set” or “primer pair” refers to a specific combination of a forward primer and a reverse primer. The “primer set” or “primer pair” may be used in a PCR reaction to generate a specific PCR product or amplicon.

In certain embodiments, the term “primer” is also intended to encompass the oligonucleotides used in ligation-mediated amplification processes, in which one oligonucleotide is “extended” by ligation to a second oligonucleotide which hybridizes at an adjacent position. Thus, the term “primer extension”, as used herein, refers to both the polymerization of individual nucleoside triphosphates using the primer as a point of initiation of DNA synthesis and to the ligation of two oligonucleotides to form an extended product.

Oligonucleotide Preparation

Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al., 1080, Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al., 1981, Tetrahedron Lett. 22:1859-1862; and the solid support method of U.S. Pat. No. 4,458,066, each incorporated herein by reference. A review of synthesis methods of conjugates of oligonucleotides and modified nucleotides is provided in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187, incorporated herein by reference.

Complementary

As used herein, “complementary” refers to a nucleic acid molecule that can form hydrogen bond(s) with another nucleic acid molecule by either traditional Watson-Crick base pairing or other non-traditional types of pairing (e.g., Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleosides or nucleotides.

Target Sequence, Region or Nucleic Acid

The terms target, “target sequence”, “target region”, and “target nucleic acid,” as used herein, are synonymous and refer to a region or subsequence of a nucleic acid which is to be amplified or detected.

Amplification Reaction

The term “amplification reaction” refers to any chemical reaction, including an enzymatic reaction, which results in increased copies of a template nucleic acid sequence or results in transcription of a template nucleic acid. Amplification reactions include reverse transcription and the polymerase chain reaction (PCR), including Real Time PCR (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Exemplary “amplification reactions conditions” or “amplification conditions” typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by a hybridization/elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a method that allows exponential amplification of short DNA sequences (usually 100 to 600 bases) within a longer double stranded DNA molecule. PCR entails the use of a pair of primers, each about 20 nucleotides in length, that are complementary to a defined sequence on each of the two strands of the DNA. These primers are extended by a DNA polymerase so that a copy is made of the designated sequence. After making this copy, the same primers can be used again, not only to make another copy of the input DNA strand but also of the short copy made in the first round of synthesis. This leads to logarithmic amplification. Since it is necessary to raise the temperature to separate the two strands of the double strand DNA in each round of the amplification process, a major step forward was the discovery of a thermo-stable DNA polymerase (Taq polymerase) that was isolated from Thermus aquaticus, a bacterium that grows in hot pools; as a result it is not necessary to add new polymerase in every round of amplification. After several (often about 40) rounds of amplification, the PCR product is analyzed on an agarose gel and is abundant enough to be detected with an ethidium bromide stain.

Real-Time or Quantitative Polymerase Chain Reaction

In other embodiments, real-time PCR, also called quantitative real time PCR, quantitative PCR (Q-PCR/qPCR), or kinetic polymerase chain reaction, is a laboratory technique based on PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. qPCRA enables both detection and quantification (as absolute number of copies of relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample. For example, in the embodiments disclosed herein, qPCR may be used to quantify the amount of fungal DNA in a patient sample. The procedure follows the general principle of PCR; its key feature is that the amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-stranded DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA. The qPCR results may be quantitated using the ΔΔCt method. This method involves calculating a ΔCt between the average target gene Ct and average housekeeping gene Ct for a given target in each treatment group. The ΔΔCt is used to calculate the “n-fold” change in gene expression between groups.

Polymerase

As used herein, a “polymerase” refers to an enzyme that catalyzes the polymerization of nucleotides. Generally, the enzyme will initiate synthesis at the 3′-end of the primer annealed to a nucleic acid template sequence. “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene 108:1), E. coli DNA polymerase I (Lecomte and Doubleday, 1983, Nucleic Acids Res. 11:7505), T7 DNA polymerase (Nordstrom et al., 1981, J. Biol. Chem. 256:3112). Thermus thermophilus (Tth) DNA polymerase (Myers and Gelfand 1991, Biochemistry 30:7661), Bacillus stearothermophilus DNA polymerase (Stenesh and McGowan, 1977. Biochim Biophys Acta 475:32), Thermococcus litoralis (Tli) DNA polymerase (also referred to as Vent DNA polymerase, Cariello et al., 1991, Nucleic Acids Res, 19: 4193, Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998 Braz J. Med. Res, 31:1239), Thermus aquaticus (Taq) DNA polymerase (Chien et al., 1976, J. Bacteoriol, 127: 1550), Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et al., 1997, Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase (Patent application WO 0132887), and Pyrococcus GB-D (PGB-D) DNA polymerase (Juncosa-Ginesta et al., 1994, Biotechniques, 16:820). The polymerase activity of any of the above enzymes can be determined by means well known in the art.

Reaction Mixture

The term “reaction mixture” as used herein, refers to a solution containing reagents necessary to carry out a given reaction. An “amplification reaction mixture”, which refers to a solution containing reagents necessary to carry out an amplification reaction, typically contains oligonucleotide primers and a DNA polymerase or ligase in a suitable buffer. A “PCR reaction mixture” typically contains oligonucleotide primers, a DNA polymerase (most typically a thermostable DNA polymerase), dNTPs, and a divalent metal cation in a suitable buffer. A reaction mixture is referred to as complete if it contains all reagents necessary to enable the reaction, and incomplete if it contains only a subset of the necessary reagents. It will be understood by one of skill in the art that reaction components are routinely stored as separate solutions, each containing a subset of the total components, for reasons of convenience, storage stability, or to allow for application-dependent adjustment of the component concentrations, and that reaction components are combined prior to the reaction to create a complete reaction mixture. Furthermore, it will be understood by one of skill in the art that reaction components are packaged separately for commercialization and that useful commercial kits may contain any subset of the reaction components which includes the blocked primers of the disclosure.

Ligation and Ligase

The term “ligation” as used herein refers to the covalent joining of two polynucleotide ends. In various embodiments, ligation involves the covalent joining of a 3′ end of a first polynucleotide (the acceptor) to a 5′ end of a second polynucleotide (the donor). Ligation results in a phosphodiester bond being formed between the polynucleotide ends. In various embodiments, ligation may be mediated by any enzyme, chemical, or process that results in a covalent joining of the polynucleotide ends. In certain embodiments, ligation is mediated by a ligase enzyme.

As used herein, “ligase” refers to an enzyme that is capable of covalently linking the 3′ hydroxyl group of neucleotide to the 5′ phosphate group of a second nucleotide. Examples of ligases include E. coli DNA ligase, T4 DNA ligase, etc.

The litigation reaction can be employed in DNA amplification methods such as the “ligase chain reaction” (LCR), also referred to as the “ligase amplification reaction” (LAR), see Barany, Proc. Natl. Acad. Sci., 88:189 (1991); and Wu and Wallace, Genomics 4:560 (1989) incorporated herein by reference. In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of the target DNA, and a complementary set of adjacent oligonucleotides, that hybridize to the opposite strand are mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, hybridization and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes. see Segev, PCT Public. No. WO9001069 A1 (1990).

Orthologs

As used herein, the term “orthologs” refers to genes in different species that apparently evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function through the course of evolution. Identification of orthologs can provide reliable prediction of gene function in newly sequenced genomes. Sequence comparison algorithms that can be used to identify orthologs include without limitation BLAST, FASTA, DNA Strider, and the GCG pileup program. Orthologs often have high sequence similarity. The present invention encompasses all orthologs of the desired protein.

Operative Associated

By “operatively associated with” is meant that a target nucleic acid sequence and one or more expression control sequences (e.g., promoters) are physically linked so as to permit expression of the polypeptide encoded by the target nucleic acid sequence within a host cell.

Percent Sequence Similarity or Percent Sequence Identity

The terms “percent (%) sequence similarity”, “percent (%) sequence identity”, and the like, generally refer to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.), etc.

To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are, or are about, of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990, 87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 1990; 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to sequences of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to protein sequences of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 1997, 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationship between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov/BLAST/ on the WorldWideWeb. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 1988; 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 1970, 48:444-453), which has been incorporated into the GAP program in the GCG software package Accelrys, Burlington, Mass.; available at accelrys.com on the WorldWideWeb), using either a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that can be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is a sequence identity or homology limitation of the invention) is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

In addition to the cDNA sequences encoding various desired proteins, the present invention further provides polynucleotide molecules comprising nucleotide sequences having certain percentage sequence identities to any of the aforementioned sequences. Such sequences preferably hybridize under conditions of moderate or high stringency as described above, and may include species orthologs.

Variant

The term “variant” may also be used to indicate a modified or altered gene, DNA sequences, enzyme, cell, etc., i.e., any kind of mutant.

Immune Response

An “immune response” refers to the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Such a response usually consists of the subject producing antibodies, B cells, helper T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest. The immune response also may include regulatory T-cells, whose activity may suppress other immune or allergic responses. In certain embodiments, “immune response” or “innate immune response” may also refer to the initial response of immune cells to the presence of microbial organism or to the presence of “pathogen associated molecular patterns (PAMPs)”, which are evolutionarily conserved amino acid or nucleic acid sequences recognized by pattern recognition receptors on immune cells. This initial response is characterized by the release of pro-inflammatory cytokines and other pro-immune mediators by the detecting immune cells and is usually necessary for the induction of the cellular and/or antibody-mediated “adaptive” immune responses discussed above. TLR ligands expressed by E. coli and Citrobacter rodentium include but are not limited to LPS (TLR4 ligand), PGN (TLR2 ligand), triacyl lipopetides (TLR2 ligand), lipoproteins (TLR2 ligand), unmethylated DNA (TLR9 ligand), and single stranded RNA (TLR7 and TLR8). Other preferred microbes for the present invention include bacterial strains selected for use as live attenuated vaccines. Examples of those include but are not limited to Mycobacterium bovis BCG vaccine against tuberculosis, Salmonella typhi Ty21a vaccine against typhoid fever, and Vibrio cholerae CVD 103-HgR vaccine against cholera. These microorganisms may additionally express the TLR5 ligand Flagellin in addition to the PAMPs shared by E. coli and Citrobacter rodentium.

Antigen and Immunogen

An “antigen” (from antibody-generating) or “immunogen” is a substance that prompts the generation of antibodies and can cause an immune response. For example, in the present invention, proteins associated with tumors may be used as antigens or immunogens to stimulate an immune response against a tumor. An “immunodominant antigen” is defined as an antigen for which a higher relative number of T cells will be specific during an immune response, compared to the numbers of T cells with T cell receptors that recognize other antigens.

Antibody

Antibodies (also knows an immunoglobulins (Ig)) are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. They are typically made of basic structural units—each with two large heavy chains and two small light chains—to form, for example, monomers with one unit, dimers with two units or pentameters with five units. Antibodies are produced by B cells. There are several different types of antibody heavy changes, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.

Although the general structure of all antibodies is very similar, a small region at the tip of the protein is extremely variable, allowing millions of antibodies with slightly different tip structures to exist. This region is known as the hypervariable region. Each of these variants can bind to a different target, known as an antigen. This huge diversity of antibodies allows the immune system to recognize an equally wide diversity of antigens. The unique part of the antigen recognized by an antibody is termed an “epitope.” These epitopes bind with their antibody in a highly specific interaction, called induced fit, which allows antibodies to identify and bind only their unique antigen in the midst of the millions of different molecules that make up an organism. Recognition of an antigen by an antibody tags it for attack by other parts of the immune system. Antibodies can also neutralize targets directly by, for example, binding to a part of a pathogen that it needs to cause an infection. Production of antibodies is the main function of the humoral immune system.

Enzyme-Linked Immunoabsorbent Assay (ELISA)

Enzyme-Linked ImmunoSorbent Assay, also called ELISA, Enzyme ImmunoAssay or EIA, is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. In ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescene ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescene. Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a “sandwich” ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though never assays employ fluorogenic substrates enabling much higher sensitivity.

Transgenic Mouse

A transgenic mouse contains additional, artifically-introduced genetic material in every cell. This often confers a gain of function, for example the mouse may produce a new protein, but a loss of function may occur if the integrated DNA interrupts another gene. A transgenic mouse is a very useful system for studying mammalian gene function and regulator because analysis is carried out on the whole organism. Transgenic mice are also used to model human diseases that involve the overexpression or misexpression of a particular protein. There are two major methods. Methods for making transgenic mice include “pronuclear microinjection”, in which the foreign DNA is introduced directly into the mouse egg just after fertilization. Using a fine needle, the DNA is injected into the large male pronucleus, which is derived from the sperm. The DNA tends to integrate as many tandemly arranged copies at a random position in the genome, often after one or two cell divisions have occurred. Therefore, the resulting mouse is only partially transgenic. If the transgenic cells contribute to the germ line, then some transgenic eggs or sperm will be produced and the next generation of mice will be fully transgenic. In another method, DNA is introduced into embryonic stem cells (ES cells). These are derived from the very early mouse embryo and can therefore differentiate into all types of cell when introduced into another embryo. DNA introduced into ES cells may integrate randomly, as in the case of pronuclear microinjection. However, if the introduced DNA is similar in sequence to part of the mouse genome, it may undergo “homologous recombination” and integrate as a single copy at a specific site. ES cells will colonize a host embryo and often contribute to the germ line, resulting in the production of some sperm carrying the extra DNA. When these transgenic sperm fertilize a normal egg, a transgenic mouse is produced with the same foreign DNA in every cell.

Knockout Mouse

A knockout mouse is a laboratory mouse in which researchers have inactivated, or “knocked out,” an existing gene by replacing it or disrupting it with an artificial piece of DNA. The loss of gene activity often causes changes in a mouse's phenotype, which includes appearance, behavior and other observable physical and biochemical characteristics. Researchers begin by harvesting embryonic stem (ES) cells from early-stage mouse embryos four days after fertilization. ES cells are used because they are able to differentiate into nearly any type of adult cell, which means that if a gene is knocked out in an ES cell, the effects can be observed in any tissue in an adult mouse. In addition, ES cells grown in the lab can be used to make knockout mice as long as 10 years after they were harvested. To produce knockout mice, researchers use one of two methods to insert artificial DNA into the chromosomes contained in the nuclei of ES cells. Both methods are carried out in vitro that is in cultured cells grown in laboratory conditions. In the first strategy, called gene targeting or homologous recombination researchers specifically manipulate a gene in the nucleus of an ES cell. Typically, this is done by introducing an artificial piece of DNA that shares identical, or homologous, sequence to the gene. This homologous sequence flanks the existing gene's DNA sequence both upstream and downstream of the gene's location on the chromosome. The cell's own nuclear machinery automatically recognizes the identical stretches of sequence and swaps out the existing gene or portion of a gene with the artificial piece of DNA. Because the artificial DNA is inactive, bearing only a genetic tag, or “reporter gene,” designed for use in tracking, the swap eliminates, or “knocks out,” the function of the existing gene.

In the second strategy, called gene trapping, researchers again manipulate a gene in an ES cell. However, instead of directly targeting a gene of interest, a random process is used. A piece of artificial DNA containing a reporter gene is designed to insert randomly into any gene. The inserted piece of artificial DNA prevents the cell's RNA “splicing” machinery from working properly, thus preventing the existing gene from producing its designated protein and knocking out its function. As in the first strategy, researchers can track the activity of the artificial reporter gene to determine the existing gene's normal pattern of activity in mouse tissues. For both gene targeting and gene trapping, the vehicle used to ferry the artificial DNA into ES cells often consists of a modified viral vector or a linear fragment of bacterial DNA. After the artificial DNA is inserted, the genetically altered ES cells are grown in a lab dish for several days and injected into early-stage mouse embryos. The embryos are implanted into the uterus of a female mouse and allowed to develop into mouse pups. The resulting mouse pups have some tissues in which a gene has been knocked out—those derived from the altered ES cells. However, they also have some normal tissues derived from the non-altered embryos into which the altered ES cells were injected. Consequently, they are not complete knockout mice. It is necessary to crossbreed such mice to produce lines of mice in which both copies of the gene (one on each chromosome) are knocked out in all tissues. These mice are referred to as homozygous knockouts.

In certain embodiments, “double knockout” mice are used. Double knockout mice have two genes that have been deleted, as described above. Examples of knockout mice of the present invention include TLR4 knockout mice (TLR4^(−/−)), IL6^(−/−), and MyD88^(−/−)/TRIF^(−/−) double knockout mice.

Pharmaceutically Acceptable

When formulated in a pharmaceutical composition, a therapeutic compound of the present invention can be admixed with a pharmaceutically acceptable carrier or excipient. As used herein, the phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are generally believed to be physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

Pharmaceutically Acceptable Derivative

The term “pharmaceutically acceptable derivative” as used herein means any pharmaceutically acceptable salt, solvate or prodrug, e.g. ester, of a compound of the invention, which upon administration to the recipient is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. Such derivatives are recognizable to those skilled in the art, without undue experimentation. Nevertheless, reference is made to the teaching of Burger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives. Preferred pharmaceutically acceptable derivatives are salts, solvates, esters, carbamates, and phosphate esters. Particularly preferred pharmaceutically acceptable derivatives are salts, solvates, and esters. Most preferred pharmaceutically acceptable derivatives are salts and esters.

Pharmaceutical Compositions and Administration

While it is possible to use a composition provided by the present invention for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent, or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Accordingly, in one aspect, the present invention provides a pharmaceutical composition or formulation comprising at least one active composition, or a pharmaceutically acceptable derivative thereof, in association with a pharmaceutically acceptable excipient, diluent, and/or carrier. The excipient, diluent and/or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The compositions of the invention can be formulated for administration in any convenient way for use in human or veterinary medicine.

Pharmaceutical Carrier

The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin (1990, Mack Publishing Co., Easton, Pa. 18042).

In one embodiment, the pharmaceutical composition is conveniently administered as a liquid oral formulation. Although there are no physical limitations to delivery of the formulation, oral delivery is preferred because of its ease and convenience, and because oral formulations readily accommodate additional mixtures, such as milk, yogurt, and infant formula. Other oral dosage forms are well known in the art and include tablets, caplets, gelcaps, capsules, and medical foods. Tablets, for example, can be made by well-known compression techniques using wet, dry, or fluidized bed granulation methods.

Such oral formulations may be presented for use in a conventional manner with the aid of one or more suitable excipients, diluents, and carriers. Pharmaceutically acceptable excipients assist or make possible the formation of a dosage form for a bioactive material and include diluents, binding agents, lubricants, glidants, disintegrants, coloring agents, and other ingredients. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used. An excipient is pharmaceutically acceptable if, in addition to performing its desired function, it is non-toxic, well tolerated upon ingestion, and does not interfere with absorption of bioactive materials.

Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit 2005). The choice of pharmaceutical excipient, diluent, and carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.

The invention also encompasses pharmaceutical compositions and vaccines. The pharmaceutical compositions and vaccine compositions of the invention include at least one of the compositions of the invention, a suitable antigen (for vaccines), and a pharmaceutically acceptable carrier or excipient. Methods of formulating pharmaceutical compositions and vaccines are well-known to those of ordinary skill in the art, as described in Remington's, supra.

Formulations

The compositions, vaccines and formulations of the present invention may comprise pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances, (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronic acid may also be used. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435 1712 which are herein incorporated by reference.

Contemplated for use herein are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which is herein incorporated by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, pellets, powders, or granules. Also, liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given by Marshall, K. In: Modern Pharmaceutical Edited by G. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated by reference. In general, the formulation will include the therapeutic agent and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.

Also contemplated for use herein are liquid dosage forms for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, and syrups, which may contain other components including inert diluents, adjuvants, wetting agents, emulsifying and suspending agents, and sweetening flavoring, coloring, and perfuming agents.

For oral formulations, the location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine, e.g., by the use of an enteric coating. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic (i.e. powder), for liquid forms a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs, or even as tablets. These therapeutics could be prepared by compression.

One may dilute or increase the volume of the therapeutic agent with an inert material. These diluents could include carbohydrates, especially mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic agent into a solid dosage form. Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab, Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, nature sponge and bentonite may all be used. The disintegrants may also be insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders, and can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants. Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the peptide (or derivative).

An artifrictional agent may be included in the formulation to prevent sticking during the formulation process. Lubricants may be used as a layer between the peptide (or derivative) and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic agent into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioetyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.

Controlled release oral formulations may be used in practicing the present invention. The therapeutic agent could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly degenerating matrices may also be incorporated into the formulation. Some enteric coatings also have a delayed release effect. Another form of a controlled release is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the therapeutic agent is enclosed in a semipermeable membrane which allows water to enter and push agent out through a single small opening due to osmotic effects.

Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan. The therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid. A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.

Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, an injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants, preserving, wetting, emulsifying, and dispersing agents. The pharmaceutical compositions may be sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured using sterile water, or some other injectable medium, immediately before use.

Vaccines

In the case of vaccines, it is often observed that a primary challenge with an antigen alone, in the absence of an adjuvant, will fail to elicit a humoral or cellular immune response. Therefore the vaccines of the invention may contain adjuvants including, but not limited to, cholera toxin, fragments and mutants or derivatives with adjuvants properties, E. coli heat-labile enterotoxin, fragments and mutants or derivatives with adjuvant properties, oil-in-water and water-in-oil emulsions, toll-like receptor ligands such as muramyl dipeptide, E. coli LPS, oiligonucleotides comprised of unmethylated DNA, poly I:C, lipoteichoic acid, peptidoglycan. Enterotoxins and their adjuvant active derivatives such as cholera toxin, heat-labile E. coli enterotoxin, pertussis toxin, shiga toxin and analogs. Other adjuvants can be used such as complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins, and potentially useful human adjuvants such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn -glycero-3-hydroxyphosphoryloxy)-ethylamine, BCG (bacille Calmette-Guerin) and Corynebacterium parvum. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed., 1984, Benjamin/Cummings: Menlo Park, Calif., p. 384). Where the vaccine is intended for use in human subjects, the adjuvant should be pharmaceutically acceptable.

Vaccine Administration

The pharmaceutical formulations and vaccines may be for administration by oral (solid or liquid), parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using ionophoresis or electroporation), transmucosal (nasal, vaginal, rectal, or sublingual), or inhalation routes of administration, or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

In a preferred embodiment, the compositions or vaccines are administered by pulmonary delivery. The composition or vaccine is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream [see, e.g., Adjei, et al. Pharmaceutical Research 1990; 7:565 569; Adjei, et al. Int. J. Pharmaceutics 1990; 63:135 144 (leuprolide acetate); Braquet, et al. J. Cardiovascular Pharmacology 1989; 13(sup5):143 146 (endothelin-1); Hubbard, et al. (1989) Annals of Internal Medicine, Vol. III, pp. 206 212 (α1 antitrypsin); Smith, et al. J. Clin. Invest. 1989; 84:1145-1146 (α 1-proteinase); Oswein, et al. “Aerosolization of Proteins”. 1990; Proceedings of Symposium on Respiratory Drug Delivery II Keystone, Colo. (recombinant human growth hormone); Debs, et al. J. Immunol. 1988; 140:3482 3488 (interferon γ and tumor necrosis factor α); and U.S. Pat. No. 5,284,656 to Platz, et al. (granulocyte colony stimulating factor). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Pat. No. 5,451,569 to Wong, et al. See also U.S. Pat. No. 6,651,655 to Licalsi et al.

Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). All such devices require the use of formulations suitable for the dispensing of the therapeutic agent. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants, surfactants and/or carriers useful in therapy. Also, the use of lipsomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

Formulations for use with a metered dose inhaler device will generally comprise a finely divided powder containing the therapeutic agent suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chloroflurocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2 tetrafluorethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the therapeutic agent, and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. The therapeutic agent should most advantageously be prepared in particulate form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal or other mucosal delivery of the therapeutic agent is also contemplated. Nasal delivery allows the passage to the blood stream directly after administering the composition to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran and saponin as an adjuvant.

The composition of vaccine of the present invention may be administered in conjunction with one or more additional active ingredients, pharmaceutical compositions, or vaccines. The therapeutic agents of the present invention may be administered to an animal, preferably a mammal, most preferably a human.

Dosage

The dosage of the therapeutic formulation or vaccine of the present invention will vary widely, depending upon the nature of the disease, the patient's medical history, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level.

Following methodologies which are well-established in the art, effective doses and toxicity of the compounds, vaccines and compositions of the instant invention, which performed well in in vitro tests, are then determined in preclinical studies using small animal models (e.g., mice or rats) in which the tumor-associated antigens, dendritic cells, polypeptides, apoptotic cells, TLR adjuvants or agonists, apoptotic cell-associated agents, pharmaceutical, or vaccine compositions have been found to be therapeutically effective and in which these drugs can be administered by the same route proposed for the human clinical trials.

For any pharmaceutical composition or vaccine used in the methods of the invention, the therapeutically effective dose can be estimated initially from animal models. Dose-response curves derived from animal systems are then used to determine testing doses for the initial clinical studies in humans. In safety determination for each composition, the dose and frequency of administration should meet or exceed those anticipated for use in the clinical trial.

As disclosed herein, the dose of the components in the compositions, vaccines and formulations of the present invention is determined to ensure that the dose administered continuously or intermittently will not exceed an amount determined after consideration of the results in test animals and the individual conditions of a patient. A specific dose naturally varies depending on the dosage procedure, the conditions of a patient or a subject animal such as age, body weight, sex, sensitivity, food, dosage period, drugs used in combination, and seriousness of the disease. The appropriate dose and dosage times under certain conditions can be determined by the test based on the above-described indices but may be refined and ultimately decided according to the judgment of the practitioner and each patients circumstances, (age, general condition, severity of symptoms, sex, etc.) according to standard clinical techniques. DC are loaded with apoptotic cells or TLR-ligand carrying apoptotic cells or apoptotic cells carrying inactivated microbes at a ratio of 1 DC to 2 apoptotic cells. DC vaccines will be administered every 28 to 30 days at 1-12×10⁶ DCs/vaccination. As a safety measure, vaccination may be initialized at 1×10⁶ DC/vaccination for the first 4 vaccines. If no toxicity is observed, after completion of 4 vaccinations, doses may be increased to 4×10⁶ DC, and finally to a maximum of 12×10⁶ DC/vaccine. These are suggested guidelines based on DC vaccinations of patients with metastatic melanoma in the study by Palucka et al. (2006) J. Immunother; 29:545-57. Actual dosage and composition or pharmaceutical formulations of TLR ligands in combination with apoptotic cell-associated agents may be determined in pre-clinical and clinical trials by standard practices known in the art.

Toxicity and therapeutic efficacy of the compositions, vaccines, and formulations of the invention can be determined by standard pharmaceutical procedures in experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio ED50/LD50. Compositions that exhibit large therapeutic indices are preferred.

The data obtained from animal studies can be used in formulating a range of doses for use in humans. The therapeutically effective doses of in humans lay preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. Ideally, a single dose of each drug should be used daily.

Kits

In one embodiment, the invention relates to a kit comprising any one of the compositions or formulations of the present invention. The compositions or formulations included in the kit may be useful for inducing T_(H)17 responses, or for inhibiting T_(H)17 responses, or for inducing T_(reg) responses. In certain embodiments, the compositions or formulations included in the kit are useful for treating a disease or condition. The kit further comprises a means for detecting improvement in the disease or condition following treatment with an agent.

The abbreviations in the specification correspond to units of measure, techniques, properties or compounds as follows: “min” means minutes, “h” means hour(s), “μL” mean microliter(s), “mL” means milliliter(s), “mM” means millimolar, “M” means molar, “μl” means micriliter(s); “mmole” means millimole(s), “kb” means kilobase, “bp” means base pair(s), and “IU” means International Units. “Polymerase chain reaction” is abbreviated PCR; “Reverse transcriptase polymerase chain reaction” is abbreviated RT-PCR; “Estrogen receptor” is abbreviated ER; “DNA binding domain” is abbreviated DBD; “Untranslated region” is abbreviated UTR; “Sodium dodecyl sulfate” is abbreviated SDS; and “High Pressure Liquid Chromatography” is abbreviated HPLC; dendritic cell is abbreviated “DC”; bone-marrow-derived dentritic cell is abbreviated “BMDC”; and culture medium is abbreviated “CM”.

EXAMPLES

The following example are included to demonstrate certain embodiments of the invention. These specific examples are described solely for purposes of illustration, and are not intended to limit the scope of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Although specific targets, terms, and values have been employed herein, such targets, terms, and values will likewise be understood as exemplary and non-limiting to the scope of this invention.

Example 1

Materials and Methods

The following describes the materials and methods employed in Examples 2-6. In all Examples, all cell culture plasticware, including culture dishes, Petri dishes, culture plates and tubes were obtained from Beckton Dickinson (BD) Falcon (Franklin Lakes, N.J.).

Description of Mice Used in Examples 2-6

The following mice were used in Examples, where described: C3H/HeOuJ mice (The Jackson Laboratory, Bar Harbor, Me.), C57BL/6J (The Jackson Laboratory, Bar Harbor, Me.), TLR4 knockout (^(−/−)) mice on the C57BL/6J background (from S. Akira, Japan), MyD88^(−/−)/Trif^(−/−)mice (from S. Akira, Japan), IL-6^(−/−) mice (on the C57BL/6J background) (from R. Medzhitov, Yale University, New Haven, Conn.). All mice were females 6-8 weeks of age. [See, Akira S and Takeda K. (2004) C R Biol. 327:581-9].

Preparation of Conditioned Media (CM) From Bone Marrow Derived Dendritic Cells (BMDC)

Bone marrow (BM)-derived GM-CSF DC cultures were grown in RPMI supplemented with GM-CSF and 5% foetal bovine serum (FBS), plus 100 μg/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 nM sodium pyruvate, 1X MEM nonessential amino acids, and 2.5 μM β-mercaptoethanol (all from Sigma-Aldrich, St. Louis, Mo.), as previously described¹. Semi-adherent cells were harvested on ice on day 5 and re-plated immediately in fresh GM-CSF medium at 1×10⁶ cells/well in 24-well tissue culture-treated plates. Soluble or phagocytic stimuli were added right away to the plates in the same medium and the cells were centrifuged for 2 min at 2000 rpm. Supernatants (conditioned medium, CM) were collected after 18 hours. LPS (from E. coli, serotype 055:B5,L-2880) was purchased from Sigma-Aldrich. BMDC were treated with various doses of soluble LPS or 2:1 A20 LPS blasts:DC to titrate the LPS such that levels of IL-6 produced by DC in response to these stimuli was similar. Soluble LPS was used at a final concentration of 1 ng/ml. The A20 B-cell line was obtained from the ATCC (TIB-208), A20 LPS blasts were prepared by culturing A20 cells at 1×10⁶ cells/mL, 3 mL/well in 6-well tissue culture plates for four days in RPMI medium supplemented with 10% FBS, with 100 μg/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 nM sodium pyruvate, plus 25 μg/mL LPS, as previously described in Blander, J. M. and Medzhitov, R. (2004) Science 304(5673):1014. Apoptosis of A20 cells and A20 LPS blasts was induced by culturing cells with 0.5 μg/mL anti-CD95 (clone Jo2; BD) for four hours. Necrotic A20 cells were prepared by submitting cells (resuspended at 2×10⁶ cells/mL in PBS) to two cycles of freezing/thawing by successive incubations in dry ice/ethanol and water at 37° C. Neutrophils and neutrophils/E. coli were prepared as follows: C57BL/6J mice were injected intraperitoneally with either 1 mL thioglycollate (Fisher) or 1 mL thioglycollate spiked with 10⁵ live DH5 E. coli (Escherichia coli K12, ATCC 23716). After 14 hours, the mice were sacrificed and cells were collected from a peritoneal wash of the contents of the abdominal cavity. Cells were centrifuged, counted, and apoptosis was induced by UV irradiation at 350 mJ. After irradiation, neutrophils were incubated for four hours at 37° C. before use as phagocytic cargo for DC. Apoptosis of A20 B-cells and neutrophils were confirmed by staining cells with cell death Annexin-V-PE detection kit (Roche, Indianapolis, Ind.) and observing a majority of Annexin-V⁺/7AAD⁻ cells after four hours. Apoptotic cells were added to BMDC at a ration of 2:1.

In Vitro T-cell Differentiation

Naïve CD4 T cells were isolated first by sorting with MACS® CD4⁺ beads (Miltenyi Biotech, Auburn, Calif.) according to manufacturer's instructions and then by fluorescence activated cell sorting (FACS) using MoFlo™, Vantage, or Influx™ cell sorter for CD25⁻CD44^(low)CD62L^(high) cells using allophycocyanin (APC)-conjugated anti-CD25 monoclonal antibody (mAb) (clone PC61.5), FITC-conjugated CD62L mAb (clone MEL-14), and PE-Cy5-conjugated CD44 mAb (clone IM7) (all from eBioscience™, San Diego, Calif.). Cells were grown in complete IMDM (Gibco, Carlsbad, Calif.) supplemented with 10% FBS, 100 μg/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 10 mM HEPES, and 1 nM sodium pyruvate, and activated on 48-well tissue-culture plates which had been coated with 4 μg anti-CD3 (clone 2C11) for 3 h at 37° C., then washed 3 times with PBS. Cultures were supplemented with 2 μg/mL anti-CD28 (clone 37-51, a kind gift from J. Allison, Sloan-Keterring Cancer Center) and anti-IL-4 ascites (clone 11B11, a kind gift from T. Moran, Mount Sinai School of Medicine, ATCC number HB188) at 1:500, mAbs 2C11 and 37-51 were purified from hybridoma supernatants by BioXCell Company (West Lebanon, N.H.). When cultured with conditioned medium (CM) from BMDC, T cells were grown in 1:1 CM:fresh complete IMDM. Cytokines and neutralizing antibodies were added at the following concentrations where indicated: IL-6 (Peprotech, Rocky Hill, N.J.) 50 ng/mL; TGF-β (Peprotech) 5 ng/mL; IL-12 (eBioscience™) 10 ng/mL; IL-23 (eBioscience™) 10 ng/mL; and-IFN-γ (clone XMG1.2, a kind gift from T. Moran) 5 μg/mL; anti-TGF-β (clone 1D11, R&D Systems, Minneapolos, Minn.) 1, 5, or 10 μg/mL; anti-IL-23p19 (clone G23-8, eBioscience™) 2, 5, 10 or 20 μg/mL; anti-IL-6 (clone MP5-20FS; eBioscience™) 1 or 5 μg/mL.

Isolation of Intraepithelial Lymphocytes (IEL) and Lamina Propria Lymphocytes (LPL)

The small intestines and colons of C57BL/6J or C3H/HeOuJ mice were harvested at the indicated times. Colons of mice of either strain that had been infected with C. rodentium wild-type or mutant strains showed inflammation grossly, with thickening of the intestinal walls and development of Peyer's patch-like tertiary lymphoid structures. To prepare IEL/LPL, small intestines and colons were flushed with HBSS medium without calcium or magnesium (Gibco). Intestines were cut longitudinally and washed twice briefly in HBSS, 2% FBS in 6-well plates and then placed in 15 mL ice-cold HBSS, 2% FBS in 50-mL conical tubes and vortexed at maximum setting for 15-20 seconds. Tissue was removed using long forceps into a new tube with 15 mL ice-cold HBSS, 2% FBS and vortexed again twice, for a total of three washes. The tissues were then placed in 50-mL conical tubes containing 25 mL HBSS, 5% FBS, and 1 mM DTT (Sigma-Aldrich). Tubes were incubated on a rocker at 37° C. for 20 minutes followed by vortexing extensively at maximum setting. For IEL, fractions were collected after two rounds of 20 minutes in HBSS/FBS/DTT. Tissues were then placed in fresh tubes containing 25 mL of PBS with 1.3 mM EDTA, incubated on a rocker for 60 minutes at 37° C. and vortexed. Tissues were then rinsed twice in RPMI, 2% FBS in 14 6-well plates, placed in new 6-well dishes with 5 mL (colons) or 7 mL (small intestines) of RPMI, 5% FBS, 1.6 mg/mL collagenase D (Roche), and cut into small pieces. Tissues were then incubated for one hour at 37° C. before homogenization using a 20 gauge syringe and filtered through a 70 μm cell strainer (BD) into a 50-mL conical tube. Wells and strainers were washed with RPMI, 5% FBS to reduce cell loss. Tubes were centrifuged to collect the cell pellet and cells were washed again in RPMI +5% FBS. Cell pellets were then resuspended in 8 mL 44% iso-osmotic Percoll™ (GE Healthcare) in RPMI and transferred to FBS-coated 15 mL polystyrene round-bottom tubes. 5 mL of 66% iso-osmotic Percoll™/RPMI was carefully layered underneath the cells layer using Pasteur pipets. Tubes were then centrifuged for 20 minutes at 2800 rpm, 4° C., with brakes in the lowest setting. After the spin, interface cells were collected using a plastic collection pipet, placed in 15-mL conical tubes, and washed twice with RPMI, 5% FBS. To restimulate the cells for measuring intracellular cytokine production, cells were then resuspended in complete IMDM with 0.1 μg/mL Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich), 0.5 μg/mL ionomycin calcium salt, from Streptomyces conglobatus (Sigma-Aldrich), and 10 μg/mL Brefeldin A, from Eupenicillium brefeldanium (Sigma-Aldrich), and cultured for four hours at 37° C. before intracellular cytokine staining.

Preparation of Tissues and Immunostaining

Colons of C3H/HeOuJ mice were harvested on day 6 and flushed with PBS. Tissues were fresh-frozen in O.C.T. Compound (Sakura Finetek, Zoeterwoude, The Netherlands) and stored at −80° C. Sections of 6 μm were cut and fixed with either 4% paraformaldehyde or ice-cold acetone for 1 h, dried, and stored at −20° C. TUNEL staining was performed using the In Site Cell Death Detection Kit, TMR Red (Roche) according to manufacturer's instructions. For immunostaining, sections were blocked and incubated with primary antibodies in a humidified atmosphere for 1 hour at room temperature. After washing, conjugated secondary antibodies were added for 35 minutes. The slides were then washed and mounted with Fluoromount-G (Southern Biotech, Birmingham, Ala.). Tissues were stained with Alexa Flur® 594-conjugated Phalloidin (Invitrogen, Carlsbad Calif.), and E. coli O antigen, polyvalent 8, O152 (Denka Seiken, Accurate Chemical US distributers, Westbury, N.Y.) [Mundy, R. et al. (2204) Infect Immun 72 (4), 2288] followed by secondary Alexa Fluor® 488 anti-rabbit IgG (Molecular Probes, Eugene, Oreg.). Analyses were performed using 10X and 20X dry objectives on a Nikon Eclipse E-600 fluorescence microscope and Adobe® Photoshop® software (Adobe Systems, San Jose, Calif.).

Citrobacter Rodentium Inuculation

Wild-type (WT) C. rodentium as well as two mutants of C. rodentium, one with a mutation in EPEC-secreted protein F (ΔEspF) [McNamara, B. P. and Donnenberg, M. S. (1998) FEMS Microbiol Lett166 (1):71], and one with a mutation in the mitochondrial associated protein (ΔMap) [Kenny, B. et al. (2002) Mol Microbiol 44 (4):1095] were obtained from B. B. Finlay, University of British Columbia, WT, ΔEspF, and ΔMap mutant C. rodentium strains exhibit similar attachment to and effacement of a colonic cell line [Crane, J. K., et al. (2001) Cell Microbiol 3 (4):197; McNamara, B. P. et al. (2001) J Clin Invest 107 (5):621], and all three stains colonize the intestinal epithelium to similar levels as shown by staining intestinal epithelium sections of infected mice for C. rodentium O antigen 01522. In addition, WT, ΔEspF- and ΔMap-infected mice all have similar numbers of C. rodentium in shed stools, and similar levels of colonic hyperplasia demonstrated by increased colon weight [Mundy, R. et al., supra].

WT and mutant C. rodentium were prepared by incubation with shaking at 37° C. for 8 hours in LB broth medium. After 8 hours, the bacterial density was assessed at an optical density of 600 nm and confirmed by plating of serial dilutions. Inoculation of mice was by oral administration with 2.5×10⁸ (for C3H/HeOuJ strain) or 2×10⁹ (for C57BL/6J strain) colony forming units (CFU). Before inoculation, mice were deprived of food and water for 8-12 hours. Tissues were collected for immunostaining and/or flow cytometry at times indicated after inoculation. Mice treated with Q-VD-OPH pan-caspase inhibitor (SM Biochemicals)⁷ received intraperitoneal injections of 0.4 mg in 15% DMSO/85% PBS at 90 minutes, 24 h, and 48 h after inoculation. C3H/HeOuJ mice also were treated on days 3 and 5 (total 5 doses) and C57BL/6J were treated on days 4, 6, and 8 (total 6 doses). Mice treated with dextran sulfate sodium (DSS) (Sigma-Aldrich) were given water containing 2.5 mg/mL DSS for seven days [Okayasu, I. et al. (1990) Gastroenterology 98 (3):694] at which point the DSS-water was removed and replaced with plain water for the reminder of the experiment. For anti-CD3-injected mice, mice were injected with 20 μg of anti-CD3 (clone 2C11) i.p. three times with an interval of two days and sacrificed four hours after the final injection [Kamanaka, M. et al. (2006) Immunity 25 (6):941]

Flow Cytometry

T cells were stimulated for four hours with 0.1 μg/mL Phorbol 12-myristate 13-acetate (PMA, Sigma), 0.5 μg/mL, ionmycin calcium salt, from Streptomyces conglobatus (Sigma-Aldrich), and 10 μg/mL Brefeldin A, from Eupenicillium brefeldanium (Sigma-Aldrich). Cells were then pelleted and resuspended in cold FACS buffer (PBS containing 0.1% NaN₃ and 1% heat-inactivated FBS). Cells were surface stained for 20 minutes at 4° C. with either: 1) APC-conjugated anti-CD4 mAb (clone GK1.5), or 2) APC-conjugated anti-CD3 mAb (clone 17A2), PE-conjugated anti-CD4 mAb (clone GK1.5), and FITC-conjugated anti-CD8α mAb (clone 53-6.7) (all from eBioscience™), in FACS buffer. Cells were then fixed and permeabilized using the IC Staining kit (eBioscience™) according to manufacturer's instructions with minor modifications (washing four times with permeabilization buffer before incubation with intracellular cytokine antibody). For Foxp3 staining in FIGS. 8 and 15, eBioscience™ Foxp3 staining kit was used according to manufacturer's instructions. Fixed and permeabilized cells were stained intracellularly with PE-conjugated anti-IL-17 mAb (clone TC11-18H10, BD, Pharmingen) or PE-conjugated anti-Foxp3 mAb (clone FJK-16s, eBioscience™), and FITC-conjugated anti-IFN-γ mAb (clone XMG1.2, BD, Pharmingen) or FITC-conjugated anti-IL-10 mAb (clone JES5-16E3, BD, Pharmingen). Samples were required on FACSCalibur™ (BD Biosciences) flow cytometer, and data analyses were conducted using FlowJo software (Tree Star, Inc., Ashland, Oreg.).

TGF-β Bioassay

Analysis of bioactive TGF-β was performed as previously described in Tesseur, I. et al. (2006) BMC Cell Biol 7: 15. MFB-F11 mouse fibroblast cells stably transfected with a reporter plasmid consisting of TGF-β-responsive Smad-binding elements coupled to a secreted alkaline phosphatase reporter gene (SBE-SEAP) were seeded at 3×10⁴ cells/well in 96-well flat bottom tissue culture plates and cultured overnight in DMEM supplemented with 10% FBS and penicillin/streptomycin (DMEM/P/S). Cells were washed twice with PBS and incubated in 50 μL serum-free DMEM for 2 hours. Conditioned medium from DC was diluted 1:5 in serum-free DMEM/P/S and 5 μL was added to the MFB-F11 cells. For detection of total TGF-β, diluted conditioned medium was activated by adding 1.5 μL 6M HCl to 150 μL of sample and incubating 10 minutes at room temperature. Neutralization was performed by adding 1.5 μL 6M NaOH. 10 μL of the cultured supernatant from MFB-F11 cells was collected after 24 hours. SEAP activity was measured using Great EscAPe™ SEAP Reporter System 3 (Clontech, Mountain View, Calif.) according to the manufacturer's protocol with 96-well white/flat bottom plates. Luciferase activity was measured with a FLUOStar Optima reader (BMG Labtech, Durham, N.C.). For each sample, the concentration of biologically active TGF-β was calculated as follows: (luciferase activity of bio-active TGF-β/luciferase activity for total TGF-β)×100.

T Cell Proliferation Assay

Day 5 WT C57BL/6J BMDC were left untreated or cultured for 6-8 hours with heat-killed C. rodentium at a multiplicity of infection (MOI) of 50. Mesenteric lymph nodes (MLN) were harvested from uninfected or infected mice that had been treated or not with Q-VD-OPH and MLN cells were isolated. These cells were incubated either with untreated BMDC in the presence or absence of 01 μg/mL anti-CD3 or with C. rodentium-exposed BMDC at two different DC:MLN cell ratios in triplicate in 96-well round-bottom plates. At 72 hours, 1 μCi of ³H-thymidine was added to each well. 18 hours after pulsing with ³H-thymidine, cells were harvested with a multiple-sample harvester and counted with a Wallac 1450 microbeta PLUS liquid scintillation counter (Perkin-Elmer, Waltham, Mass.).

RNA Isolation cDNA Synthesis and Quantitative Real-Time RT-PCR

T cells cultured for 48 hours under the indicated conditions were lysed and RNA isolated by TRIzol® extraction (Invitrogen) according to manufacturer's instructions. For restimulated T cells cultures, cells were collected after four days and plated on new plates coated with anti-CD3 in the presence or absence of 20 ng/mL recombinant (rm) IL-23 (eBioscience™) for six hours. RNA isolation was performed in the same manner as above. Quantitative real-time, reverse transcription polymerase chain reaction (qPCR) was conducted on an ABI Prism® 7900 instrument (Applied Biosystems, Foster City, Calif.) with primer pairs and probes as follows. All probe sequences are in the format: 5′ FAM-sequence-BHQ-1 3′:

(HPRT Probe, SEQ ID NO: 61) TGTTGGATACAGGCCAGACTTTGTTGGAT (HPRT FW, SEQ ID NO: 62) CTGGTGAAAAGGACCTCTCG (HPRT RV, SEQ ID NO: 63) TGAAGTACTCATTATAGTCAAGGGCA (β-actin Probe, SEQ ID NO: 64) AGCCACCCCCACTCCTAAGAGGAGG (β-actin FW, SEQ ID NO: 65) GAAGTCCCTCACCCTCCCAA (β-actin RV, SEQ ID NO: 66) GGCATGGACGCGACCA (IL-6 Probe, SEQ ID NO: 67) TCTGCAAGAGACTTCCATCCAGTTGCCT (IL-6 FW, SEQ ID NO: 68) CCAGAAACCGCTATGAAGTTCC (IL-6 RV, SEQ ID NO: 69) TCACCAGCATCAGTCCCAAG (IL-17A Probe, SEQ ID NO: 70) TCTGGGAAGCTCAGTGCCGCCACCAGC (IL-17A FW, SEQ ID NO: 71) CTCCAGAAGGCCCTCAGACTAC (IL-17A RV, SEQ ID NO: 72) AGCTTTCCCTCCGCATTGACACAG (Foxp3 Probe, SEQ ID NO: 73) ATCCTACCCACTGCTGGCAAATGGAGTC (Foxp3 FW, SEQ ID NO: 74) CCCAGGAAAGACAGCAACCTT (Foxp3 RV, SEQ ID NO: 75) TTCTCACAACCAGGCCACTTG (RORγt Probe, SEQ ID NO: 76) AAGGGCTTCTTCCGCCGCAGCCAGCAG (RORγt FW, SEQ ID NO: 77) CCGCTGAGAGGGCTTCAC (RORγt RV, SEQ ID NO: 78) TGCAGGAGTAGGCCACATTACA (Tbet Probe, SEQ ID NO: 79) CCGGGAGAACTTTGAGTCCATGTACGC (Tbet FW, SEQ ID NO: 80) CAACAACCCCTTTGCCAAAG (Tbet RV, SEQ ID NO: 81) TCCCCCAAGCAGTTGACAGT (IL-22 Probe, SEQ ID NO: 82) TGAGCACCTGCTTCATCAGGTAGCA (IL-22 FW, SEQ ID NO: 83) TCCGAGGAGTCAGTGCTAAA (IL-22 RV, SEQ ID NO: 84) AGAACGTCTTCCAGGGTGAA (IL-12p40 Probe, SEQ ID NO: 85) TGCAGCAAGTGGGCATGTGTTCC (IL-12p40 FW, SEQ ID NO: 86) CTCAGGATCGCTATTACAATTCCTC (IL-12p40 RV, SEQ ID NO: 87) TTCCAACGTTGCATCCTAGGATC (IL-12p35 Probe, SEQ ID NO: 88) TCTGGCCGTCTTCACCATGTCA (IL-12p35 FW, SEQ ID NO: 89) CTTAGCCAGTCCCGAAACCT (IL-12p35 RV, SEQ ID NO: 90) TTGGTCCCGTGTGATGTCT

The above primers and probe sets synthesized by Biosearch Technologies (Novato, Calif.). Applied Biosystems TaqMan® gene expression assay ID numbers Mm00518984_ml and Mm00770031_ml were used for the primers and probe sets for IL-23p19 and tumor necrosis factor (ligand) superfamily member 15 (TNFsf15), respectively. The sequences of these commercially-available primers are proprietary and are not disclosed by Applied Biosystems. All primers annealed in different exons and when possible, probes were designed to anneal across exon boundaries. Sequence references where the sequences are based on a previously published sequence are as follows: Rorγt and IL-17A [Ivanov, II et al. (2006) Cell 126 (6):1121], IL-6 [Wang, T. et al., (2004) Nat Med 10 (12):1366], IL-22 [Zheng, Y. et al. (2008) Nat Med 14 (3):282], Foxp3 [Uhlig, H. H. et al (2006) J Immunol 177 (9):5852].

Quantitative PCR was performed using TaqMan® quantitative PCR Master Mix at a concentrate of 1X (Applied Biosystems). Reactions were run in duplicates and samples were normalized to the internal controls β-actin and HPRT. “Fold inductions” were calculated using the ΔΔCt method relative to T cells activated under neutral conditions (No cytokines or CM).

Enzyme-Linked Immunoabsorbent Assays (ELISA)

Supernatant from cultured DC or T cells was collected at the times indicated. ELISA monoclonal Ab (mAb) pairs used were as noted below in Table 1. All mAbs were obtained from BD, Pharmingen except the IL-23p19 capture antibody, clone 5B2, from eBioscience™. All ELISA antibodies were used at 1.5 μg/mL capture and 1.5 μg/mL detection with the following exceptions: IL-23p19 capture mAb was used at 4 μb/mL, IL-10 capture and detection mAbs were used at 2 μg/mL.

TABLE 1 ELISA Antibody Pairs Cytokine Capture Ab Biotinylated Ab TGF-β A75.2 A75.3 IL-6 MP5-20F3 MP5-32C11 IL-12p40/p70 C15.6 C17.8 IL-23p19 5B2 C17.8 IL-17 TC11-18H10 TC11-BH4.1 IL-10 JES5-2A5 SXC-1

Example 2

DC Cytokine Production Following Concomitant Exposure to LPS and Apoptotic Cells

The following example demonstrates the cytokines produced by DC following exposure to various inflammatory stimuli and the ability of treated DC to stimulate T_(H)17 cell differentiation.

It was next determined which innate immune recognition events trigger simultaneous synthesis of IL-6 and TGF-β. Whereas phagocytosis of apoptotic cells induces TGF-β synthesis by macrophages⁷. IL-6 synthesis is strongly induced in innate immune cells when microbial structures activate pattern recognition receptors like Toll-like receptors (TLRs)^(6, 14). However, it was unknown whether concomitant litigation of TLRs during phagocytosis of infected apoptotic cells might constitute a scenario where IL-6 and TGF-β may be induced together.

In these experiments, either apoptotic neutrophils isolated from the peritoneal cavity of mice following injection of live E. coli (Apop./Neutrophils/E. coli), or apoptotic B-cells carrying the TLR4 ligand lipopolysaccharide (LPS) generated from LPS-treated B-cells (ApopA20/LPS-blasts)¹⁵ were used. As controls for the requirement for a microbe or TLR adjuvant for T_(H)17 induction, either apoptotic neutrophils (Apop./Neutrophils) or apoptotic A20 B cells (Apop./A20) were used. Bone marrow DC were stimulated with LPS or phagocytic cargo as indicated in FIG. 1 and cytokine production was measured by ELISA in culture supernatants following 18 hours of culture. DC that phagocytosed apoptotic LPS-blasts secreted more TGF-β and IL-23 than DC treated with free LPS and similar amounts of IL-6 where the concentration of free LPS chosen approximated IL-6 levels induced by apoptotic LPS-blasts (FIG. 1). Importantly, phagocytosis of apoptotic cells and apoptotic LPS-blasts uniquely induced production of biologically active TGF-β whereas free LPS did not (FIG. 1). Notably, despite similar IL-6 levels, the level of IL-12 produced by DC following phagocytosis of apoptotic LPS B cell-blasts was consistently lower than that in response to free LPS (FIG. 1), which may also favor T_(H)17 over T_(H)1 development.

The mRNA expression of DC stimulated in vitro by the stimuli indicated in FIG. 2 was determined by quantitative reverse transcription-polymerase chain reaction (qPCR) analyses. Analysis by qPCR of RNA from these DC further showed induction of IL-6, IL-23p19, IL-12p35, IL-12p40, IL-10, and TNF family member, TL1A (Tnfsfl5), which promotes T_(H)17 proliferation¹⁶, in response to apoptotic LPS-blasts and apoptotic neutrophils/E. coli. For all cytokines tested, the levels of induction were similar to levels induced by free LPS (FIG. 2). These transcripts were also induced in DC in response to infected apoptotic neutrophils, but not to uninfected apoptotic B-cells or uninfected apoptotic neutrophils (FIG. 2). The raw data was normalized to β-actin and HPRT and expressed as fold-induction over unstimulated DC.

Example 3

T_(H)17 and T_(reg) Cell Differentiation

The following example describes whether the cytokine milieu created by the conditions of DC activation described in Example 2, above, is conducive for T_(H)17 induction.

Naïve (CD25⁻CD62L^(high)CD44^(low)) CD4 T-cells, isolated from C57BL/6J mice and activated with 4 μg anti-CD3 and 2 μg/mL anti-CD28 plus anti-IL-4, were cultured for three days in conditioned media (CM) derived from DC that phagocytosed apoptotic LPS-blasts or apoptotic B cells (control) with or without IFN-γ neutralizing antibody. Following restimulation with PMA and ionocmycin, the CD4 T-cells cultured with DCCM-apoptotic LPS-blasts secreted IL-17, as determine by ELISA of the culture supernatants, strongly suggesting differentiation into the T_(H)17 lineage (FIG. 3, filled bars).

Standard in vitro protocols for generating T_(H)17 require neutralizing the antagonistic effects of the cytokine interferon-γ (IFN-γ)^(1, 10, 11), and TLR ligands are classically considered to induce T_(H)1 cells producing IFN-γ⁶. Neutralization of this cytokine allowed some induction of IL-17 secreting (IL-17) CD4 T-cells by CM from DC treated with free LPS (FIG. 3, open bars). In contrast, anti-IFN-γ was not required for IL-17 production by T-cells activated in DCCM-apoptotic LPS-blasts (FIG. 3, open bars), indicating that DC stimulation by TLR ligands within the context of apoptotic cells creates particularly efficient conditions, which more closely mimic physiological settings, for the generation of IL-17⁺ CD4 T-cells. No IL-17 was detected when naïve CD4 T-cells were cultured in the presence of CM from DC that phagocytosed apoptotic B-cells not carrying TLR ligands (DCCM-apoptotic B-cells), and supplementation of this CM with IL-6 restored IL-17 production, indicating the presence of TGF-β and reinforcing the contribution of TLRs to IL-6 production (FIG. 3, filled bars). Here, anti-IFN-γ consistently increased the levels of IL-17 (FIG. 3, open bars).

Moreover, qPCR performed with RNA from sorted naïve CD4 T-cells after 48 hours of culture with DCCM from the indicated groups. qPCR analysis showed high induction of the T_(H)17 lineage specific transcription factor RORγt⁴ (Rorc) and IL-17A when these cells were activated in the presence of DCCM-apoptotic LPS-blasts, and DCCM-apoptotic B-cells supplemented with IL-6 (FIG. 4). Notably, induction of the IL-10 family cytokine, IL-22, synthesis of which is induced by IL-23¹⁷, was greatest in response to DCCM-apoptotic LPS-blasts (FIG. 4), consistent with the ability of apoptotic LPS-blasts, but not apoptotic cells, to induce IL-23 cytokine synthesis (FIG. 1). IL-22 was not induced upon stimulation with TGF-β plus IL-6, likely due to the absence of IL-23 under these conditions (FIG. 4). As expected, none of the T-cells, except the ones activated in the presence of IL-12, expressed the T_(H)1 lineage-specific transcription factor T-bet¹⁸ (FIG. 4). Collectively, these results confirmed that IL-17³⁰ CD4 T-cells generated in response to phagocytosis of TLR ligand-containing apoptotic cells were bona fide T_(H)17 cells.

Because development of T_(reg) cells is reciprocally related to T_(H)17 cells¹⁰ expression of Foxp3, a transcription factor unique to T_(reg) cells¹⁹ was examined. After 3 days of culture with DCCM from the indicated groups, T cells were restimulated with PMA and ionmycin for 4 hours with Brefeldin A before intracellular cytokine staining for IL-17 and IFN-γ was performed, and expression of these cytokines was determined by FACS. In FIG. 5, FACS plots are gated on CD4⁺ cells and quadrant percentiles of cells staining positive for the indicate cytokines are shown.

IL-17⁺ CD4 T-cells developed in DCCM-apoptotic LPS-blasts and DCCM-apoptotic neutrophils/E. coli and, as expressed, did not express Foxp3 (FIG. 5). In contrast, Foxp3-expressing cells were generated in response to DCCM-apoptotic B-cells or DCCM-apoptotic neutrophils. No IL-17 producing cells were obtained under these conditions, indicating preferential T_(reg) development (FIG. 5). Addition of IL-6 to DCCM-apoptotic B-cells or to DCCM-apoptotic neutrophils restored IL-17 production and markedly impaired Foxp3 expression (FIG. 5). Quantitative RT-PCR showed Foxp3 induction inversely mirrored RORγt, with highest induction when T-cells were activated in the presence of DCCM-apoptotic B-cells or TGF-β (FIG. 4). Foxp3 induction was impaired when IL-6 was added consistent with the ability of IL-6 to inhibit Foxp3 induction¹⁰, and when T-cells were activated in the presence of DCCM-apoptotic LPS-blasts (FIG. 4), which contains IL-6 (FIG. 1). Although Foxp3 induction was not completely abrogated under the latter two conditions, this had no negative impact on T_(H)17 development (FIGS. 3 and 5) perhaps owing to the high levels of RORγt induced under these two conditions (FIG. 4, RORc panel). Persistence of Foxp3 transcripts in the TGF-β plus IL-6 conditions (unlike previous reports^(10, 20)) may be a consequence of the comparatively high levels of TGF-β (5 ng/ml) that were used. This concentration was chosen as it approximates the average concentrations of TGF-β present within DCCM-apoptotic B-cells, or DCCM-apoptotic LPS-blasts (FIG. 1). These data collectively demonstrate that phagocytosis of apoptotic cells by DC instructed development of Foxp3 expressing T_(reg) cells and phagocytosis of infected apoptotic cells (which have TLR ligands) instructed development of T_(H)17 cells.

FIG. 6 shows that the absence of TLR signaling in DC impairs T_(H)17 cell development in response to phagocytosis of infected apoptotic cells, and support T_(reg) development instead. Naïve CD4 T cells were isolated from C57BL/6J mice and activated with 4 μg anti-CD3 and 2 μg/mL anti-CD28 plus anti-IL-4 with conditioned medium from wild-type C57BL/6J, Myd88^(−/−)/Trif^(−/−), or Tlr4^(−/−) BMDC under the indicated conditions. IL-6 and TGF-β cytokines were added to T cell—DC conditioned medium cultures. Cells were recovered after three days and surface staining for CD4 and intracellular cytokine staining for Foxp3 were performed. Cells were analyzed for flow cytometry and in the plots shown in FIG. 6, plots are gated on CD4⁺ cells and quadrant percentiles of cells staining positively for the indicated cytokines or markers are shown. Under these conditions, CM from DC derived from Myd88^(−/−) Trif^(−/−) mice (FIG. 6, where absence of the signaling adaptors MyD88 and Trif abrogates responses through all TLRs¹⁴) or Tlr4^(−/−) mice (FIG. 6, where absence of TLR4 abrogates responses to LPS¹⁴) did not support T_(H)17 development, but rather supported development of Foxp3 express T_(reg) cells.

FIG. 7 demonstrates that phagocytosis of necrotic cells by BMDC does not induce T_(H)17 differentiation. Naïve CD4 T cells were isolated from C57BL/6J mice and activated with 4 μg anti-CD3 and 2 μg/mL anti-CD28 plus anti-IL-4 with conditioned medium from C57BL/6J BMDC that phagocytosed either apoptotic A20 LPS blasts, apoptotic A20 cells, or necrotic A20 cells. Cells were recovered after three days and restimulated with PMA plus ionomycin with Brefeldin A before intracellular cytokine staining for IL-17 and IFN-γ, and analysis by flow cytometry. Plots were gated on CD4+ cells and quadrant percentiles of cells staining positively for the indicated cytokines are shown.

The TLR ligand and apoptotic cell cannot be administered as separate entities as this combination does as such does not lend to the induction of T_(H)17 responses. The TLR ligand must be physically incorporated and present within the apoptotic cell in order to effectively mimic an infected apoptotic cell and induce a T_(H)17 response. See Example 5 and FIG. 25 below.

Next, naïve CD4 T-cells were isolated from C57BL/6J mice and activated with 4 μg anti-CD3 and 2 μg/ML anti-CD28 plus anti-IL-4 with CM from C57BL/6J (WT) BMDC that had phagocytosed apoptotic LPS B cell-blasts. Neutralizing mAbs were added (or not) as indicated (anti-TGF-β 1 μg/mL, 5 μg/mL, 10 μg/mL; anti-IL-23p19 5 μg/mL, 10 μg/mL, 20 μg/mL; anti-IL-6 1 μg/mL, 5 μg/mL). After four days, cells were re-plated on anti-CD3-coated plates for 48 h. Cytokines were quantified in supernatants by ELISA. As shown in FIG. 8, development of T_(H)17 cells in the presence of DCCM-apoptotic LPS-blasts were severely compromised in a dose dependent manner in the presence of neutralizing antibody to TGF-β, with no further synergistic inhibition with a neutralizing antibody to the p19 polypetide of IL-23 (FIG. 8). Neutralization of p19 alone still permitted the existence of a fraction of T_(H)17 cells, consistent with the role of IL-23 in expanding but not initiating development of these cells (FIG. 8). Neutralization of IL-6 also strongly inhibited IL-17 secretion (FIG. 8). Its requirement was further documented by the fact that IL-6^(−/−)DCCM-apoptotic LPS-blasts failed to support development of T_(H)17 cells (FIG. 9). Addition of exogenous IL-6 to DCCM-apoptotic B-cells from both WT and IL-6^(−/−)DC, led to similar levels of IL-17 production from activated naïve CD4 T-cells (FIG. 9). FIG. 10 shows the levels of IL-6 and IL-12 produced by CD4 T cells following culture with DCCM treated with the conditions shown (resting, LPS, apoptotic B cells, or LPS blasts) in WT or IL-6^(−/−) mice. IL-6^(−/−) failed to produce IL-6, but produced similar levels of IL-12 following exposure to LPS or apoptotic A20 LPS blasts. Collectively, these data suggest that DC phagocytosis of infected apoptotic cells results in production of cytokines strongly conducive for T_(H)17 differentiation.

Example 4

Role of Toll-like Receptor Signaling in T_(H)17 Development

The following example describes whether development of IL-17⁺ CD4 T-cells required TLR signaling within DC upon phagocytosis of infected apoptotic neutrophils or apoptotic LPS-blasts.

Under the same conditions as in Example 3, CM from DC derived from Myd88^(−/−)Trif^(−/−) mice (FIG. 11, where absence of the signaling adaptors MyD88 and Trif abrogates responses through all TLRs¹⁴) or Tlr4^(−/−) mice (FIG. 12, where absence of TLR4 abrogates responses to LPS¹⁴) did not support T_(H)17 development. However, consistent with the lack of involvement of TLRs in recognition of apoptotic cells per se, generation of T_(H)17 cells was unaffected by the absence of TLR signaling under conditions when DCCM-apoptotic B-cells or DCCM-apoptotic neutrophils were supplemented with Il-6 (FIGS. 11 and 12). Furthermore, DC derived from MyD88^(−/−)Trif^(−/−)and Tlr4^(−/−) mice were able to direct induction of IL-17⁺ CD4 T-cells when stimulated with curdlan, a fungal β-glucan that activates DC independently of TLR4, Trif or MyD88, and induces T_(H)17 development when TGF-β is concomitantly present²¹.

Example 5

Induction of IL-10 Regulatory Component

The following example describes the ability of TLR ligands and apoptotic cells to induce IL-10 secreting, “regulatory” T cells in addition to T_(H)17 cells.

Given the reported implications of T_(H)17 cells in autoimmunity, and the present finding that infected apoptotic cells can drive T_(H)17 development, it was next asked whether this process is accompanied by the induction of a regulatory component that might curb the pathogenic potential of these T-cells. Naïve CD4 T-cells were isolated from C57BL/6J mice and activated with 4 μg anti-CD3 and 2 μg/mL and anti-CD28 plus anti-IL-4 with CM from C57BL/6J, Myd88^(−/−)Trif^(−/−) or Tlr4^(−/−) BMDC under the indicated conditions. For the data shown in FIGS. 13, 17 and 24, after three days, cells were restimulated with PMA and ionmycin for four hours with Brefeldin A before intracellular cytokine staining for IL-17 and IFN-γ, or IL-17 and IL-10, and analyzed by flow cytometry. Plots were gated on CD4⁺ cells and quadrant percentiles of cells staining positively for the indicated cytokines are shown.

It was found that 25% of the IL-17³⁰ CD4 T-cells induced by DCCM-apoptotic LPS-blasts, also secreted IL-10, a potent anti-inflammatory cytokine²² (FIG. 13) (for IFN-γ, IL-17 and Foxp3 profiles, see FIG. 24). Notably, a distinct IL-10 secreting (IL-10⁺) CD4 T-cell population that did not secrete IL-17 was also induced. Similar ‘IL-10⁺ only’ cells and ‘dual IL-10⁺ and IL-17^(+’) cells were induced in response to DCCM-apoptotic B-cells supplemented with IL-6 (FIG. 13). It was found that induction of IL-10 transcripts in response to DCCM-apoptotic B-cells, although by staining IL-10⁺ CD4 T-cells could not be detected in response to apoptotic cells alone, perhaps due to differences in the kinetics of expression (FIG. 13). Consistent with previous reports^(23,24), TGF-β plus IL-6 also induced three distinct populations: ‘IL-17⁺ only’, ‘IL-10^(+’) only, and ‘dual IL-10⁺ and IL-17^(+’) CD4 T cells (FIG. 13).

In some experiments, after four days, cells were re-plated on anti-CD3-coated plates for 48 h in the presence or absence of 10 ng/mL IL-23. Re-stimulation of these cultured CD4 T-cells in the presence of IL-23 only slightly increased the levels of IL-17 produced, consistent with IL-23's primary role in expanding T_(H)17 cells¹⁰⁻¹² (FIG. 14, top panel). However, the total levels of IL-10 produced were markedly impaired, as previously reported²³, when IL-23 was present upon re-stimulation (FIG. 14, bottom panel). In contrast to IL-17, 30-45% of IL-10 secretion remained in response to DCCM-apoptotic LPS-blasts or DCCM-neutrophils/E. coli when TLR signaling was absent in the phagocytic DC (FIGS. 15 and 16), and was confined to the ‘IL-10⁺ only’ cells (FIG. 17). As expected, all three populations were intact in response to MyD88^(−/−)Trif^(−/−)DCCM-apoptotic+IL-6 compared to WT DCCM-apoptotic+IL-6 (FIG. 17). These data collectively suggest that the initial development of IL-17 secreting cells in response to apoptosis of infected cells is accompanied by concomitant induction of IL-10 secreting populations. IL-10 production may serve to limit excessive inflammatory responses mediated by those cells during infections²². Moreover, the inflammatory cytokines prevailing upon reactivation of these cells may directly impact their IL-10-mediated rather than IL-17mediated effector functions.

In one set of experiments, the results of which are shown in FIG. 25, naive CD4 T cells were isolated from C57BL/6J mice and activated with 4 μg anti-CD3 and 2 μg/mL anti-CD28 plus anti-IL-4 with CM from wild-type C57BL/6J BMDC under various conditions, as indicated in the Figure. When apoptotic cells and LPS were co-administered, but as separate entities (Apoptotic A20+LPS), a surprising result was found. CM derived from DC stimulated with LPS during phagocystosis of apoptotic cells induced neither IL-17 nor IL-10 secreting cells despite the expectation that LPS would induce DC production of IL-6 (FIG. 25). However, when DC were stimulated with apoptotic A20/LPS blasts (where the apoptotic cell and TLR ligand are physically associated). CM derived from these cultures induced IL-17 and IL-10 secreting CD4 T cells (FIG. 25). These results demonstrated that the TLR ligand and apoptotic cell should be delivered to DC as one entity (which allows them to be internalized together by a DC. These results also strengthened the discovery of the present invention that an infected apoptotic cell is uniquely capable of inducing T_(H)17 responses and that TLR ligands must be physically present within the apoptotic cell to accurately mimic an infected apoptotic cell. These results also demonstrated that the T_(H)17-inducing effects of A20/LPS blasts were not due to contaminating free LPS. Furthermore, CM from cultures of DC stimulated with apoptotic A20 cells and IL-6 (Apoptotic A20+IL-6) stimulated IL-17 and IL-10 secreting CD4 T cells, indicating that addition of IL-6 can overcome the need for the presence of a TLR ligand (or adjuvant) associated as a single entity with an apoptotic cells for the induction of a T_(H)17-inducing DC.

Example 6

Blockade of Apoptosis

The following example describes whether blockade of apoptosis impairs development of T_(H)17 cells in vitro during bacterial infections known to trigger T_(H)17 responses.

Citrobacter rodentium is a rodent pathogen that serves as a model for human infections with the attaching and effacing enteropathogenic and enterohemorrhagic E. coli ⁹. C3H/HeOuJ mice were infected orogastrically with C. rodentium wild-type (WT), C. rodentium ΔEspF, or C. rodentium ΔMap, and/or treated with 20 mg/kg caspase inhibitor Q-VD-OPH intraperitoneally on days 0, 1, 2, 3, and 5. Mice were sacrificed on day 6 and colons were harvested and frozen for tissue sectioning and TUNEL analysis.

FIG. 18 shows the inhibition of apoptosis by pan-caspase inhibitor Q-VD-OPH. A20 cells were incubated in the presence or absence of anti-Fas (anti-CD95) to induce apoptosis with or without the indicated concentrations of Q-VD-OPH for four hours before staining for Annexin-V and 7-AAD and analysis by flow cytometry. When A10 LPS blasts were incubated with Q-VD-OPH during Fas treatment, they were protected from undergoing Fas-induced apoptosis as shown by Annexin V/7AAD staining. In the absence of Q-VD-OPH treatment, 100% of the cells become apoptotic at later time points.

Importantly, orogastric infection with this pathogen resulted in massive apoptosis in intestinal epithelial cell^(25, 26), as measured by TUNEL staining and quantified in the graph in FIG. 19. Moreover, IL-17⁺ CD4 T-cells were increased in number and predominate within the lamina propria^(11, 17) (LP) of C. rodentium infected mice (shown in FIG. 20), to a lesser degree in intraepithelial lymphocytes and Peyer's patches, and not detectable in the mesenteric lymph nodes (FIG. 23) and spleen. Such infection also led to increased numbers of IL-17⁺ CD4 T-cells in the small intestinal and colonic LP of the more susceptible C3H/HeOuJ mice²⁶ (FIG. 21).

Treatment of C. rodentium infected mice with the pan-caspase inhibitor Q-VD-OPH resulted in decreased numbers of TUNEL cells (FIG. 19) and also profoundly diminished the number of IL-17⁺ CD4 T-cells in both strains of infected mice, and in C57BL/6J mice to levels similar to those found in uninfected mice (FIGS. 20 and 21). Thus, caspase inhibition results in blockade of epithelial cell apoptosis in a relevant infection model in vivo, and profoundly interferes with the generation of a T_(H)17 response.

Given that Q-VD-OPH is a broad-spectrum caspase inhibitor, it likely affects processes independent of apoptosis, like activation of the inflammasome, which is important for generation of immune responses²⁷. Secretion of cytokines such as IL-1 and IL-18 may thus be perturbed by Q-VD-OPH. Therefore, the requirements for apoptosis in T_(H)17 induction was tested by a fundamentally different approach, based on the usage of a C. rodentium mutant incapable of inducing apoptosis. EspF (EPEC-secreted protein F) and Map (mitochondrial associated protein) are effectors encoded by the locus of enterocyte effacement (LEE) pathogenicity island²⁸. Despite similar colonization (FIG. 19), shedding in the stool, and colonic hyperplasia, only WT and ΔMap induce apoptosis and tight junction disruption in vitro and in vivo, while ΔEspF mutants fail to do so^(25, 26, 29, 30). This is indicated by the increased numbers of apoptotic TUNEL⁺ cells in the distal colon of WT- and ΔMap-infected mice, but not ΔEspF-infected compared to uninfected mice (FIG. 19). Thus, development of T_(H)17 cells in the intestinal LP in response to infection with these strains of C. rodentium was assessed. Equivalent numbers of CD4 and CD8 T-cells were present in the LP of all WT-, ΔEspF- and ΔMap-infected C57BL/6J (FIG. 20, leftmost panels) and C3H/HeOuJ mice. Compared to uninfected mice, modest increases were observed in IL-17⁺ CD4 T-cells in the small intestinal LP in response to WT and ΔMap infection, and not in response to ΔEspF infection (FIG. 20 and 21, small intestine panels). WT and ΔMap infection induced larger increases in the IL-17³⁰ CD4 T-cells in the colonic LP, but notably the numbers of IL-17⁺ CD4 T-cells in ΔEspF-infected mice were similar to those in uninfected controls (FIGS 20 and 21, colon panels). In contrast to IL-17, similar levels of IFN-γ CD4 T-cells were found in the small intestinal LP of C57BL/6J mice infected with WT, ΔEspF, and ΔMap C. rodentium (FIG. 20, small intestine panels) consistent with intact expression of the bacterial outer membrane protein, intimin, reported to drive T_(H)1 responses in C. rodentium infections^(9, 11).

Because ΔEspF can neither induce apoptosis nor disrupt tight junctions, it remained possible that tight junction disruption per se could drive the observed T_(H)17 response. However, tight junction disruption should not be affected in Q-VD-OPH treated WT C. rodentium infected mice as Q-VD-OPH despite reducing apoptosis, has no effects on infection induced barrier function³⁰. Furthermore, dextran sulfate sodium (DSS)-induced damage of the intestinal epithelium did not increase IL-17⁺ CD4 T-cells over uninfected controls (FIG. 22). For DSS experiments, C57BL/6 mice treated with DSS and sacrificed on day 9. Lamina propria lymphocytes (LPL) were isolated from small intestines (sm. Intest) and colons. LPL were the restimulated with PMA and ionomycin for 4 hours with Brefeldin A before surface staining for CD4 and intracellular cytokine staining for IL-17 and IFN-γ, and analysis by flow cytometry. Plots were gated on CD4⁺ cells and quadrant percentiles of cells staining positively for the indicated cytokines are shown. These results argue against a role for disruption of intestinal epithelium integrity as a stimulus for T_(H)17 development.

Example 7

TLR Ligand-carrying or Infected Apoptotic Cell Engineered to Express an Exogenous Immune Antigen

Generation of TLR ligand-carrying or infected apoptotic cell engineered to express an exogenous immune antigen derived of microbial or host origin is desirable for inducing a T_(H)17 immune response directed against a tumor-associated immune antigen as a form of tumor immunotherapy, or for inducing a T_(H)17 immune response directed against a particular immunodominant antigen expressed by a given bacterium. Such apoptotic cells may be prepared, by way of non-limiting example, by the following method.

A 1) A cell line is transfected with the exogenous immune antigen of interest (e.g., a tumor-associated antigen, an immunodominant antigen associated with an autoimmune disease or chronic inflammatory disease). This cell line can be a phagocytic cell line, such as one of monocytic or macrophage origin. The antigen may be a tumor-associated antigen, for example where T_(H)17 immune responses are desired against a given tumor, or an immunodominant antigen derived from a bacterium. This antigen can be expressed from a recombinant mammalian expression vector or viral vector engineered to encode the game sequence for the antigen. This gene sequence can either be the full gene or a portion of it encompassing immunodominant regions, if these are known. Clones derived from this transfected cell line are then propagated where they stably express the exogenous immune antigen at high levels. Alternatively, a tumor cell line is chosen that represents the tumor in the mammal. This tumor cell line would naturally express the tumor-associated antigen.

2) TLR ligands are introduced into the cell line. For non-phagocytic cell lines, a TLR ligand such as CpG or Poly (I:C) may be electroporated into the cell line. For phagocytic cell lines (e.g., a macrophage or monocytic cell line), an innoocuous microbe such as E. coli K12 may be given to the cells at a ratio of 10 microbes to one cell. Alternatively, attenuated Mycobacterium bovis BCG strain may be used, for example. For safety, the innocuous microbe is further inactivated by exposure to heat, UV, or fixative. Viable microbes are not necessary to induce the desired T_(H)17 response. The microbe here serves only to provide a source of mixed TLR ligands.

A variation of the above method is to choose a phagocytic cell line to which an inactivated form of the infecting microbe is given. In this variation, step 1 above is eliminated. For example, if T_(H)17 responses against a respiratory infection with Klebsiella pneumoniae are desired, a phagocytic monocytic cell line is given heat-inactivated K. pneumonia at a ratio of 10 bacteria to one cell for a period of 30-45 minutes. The cells would then internalize the inactivated K. pneumonia thus in effect carry not only the TLR ligands derived from K. pneumonia, but all the exogenous antigens derived from K. pneumonia as well. The cells are then washed to remove excess extracellular K. pneumonia that may not have been internalized.

3) Apoptosis is induced in the transfected cell line or tumor cell line now carrying TLR ligands. Alternatively, apoptosis is induced in the phagocytic cell line following its internalization of the desired microbe. The trigger chosen to induce apoptosis depends on the cell type. This trigger can be UV irradiation at a dose sufficient to induce Annexin-V⁺ 7-ADD⁻ at early time points (4 hours). High doses of UV should be avoided as these induce necrosis (which does not induce T_(H)17 responses) measured by the appearance of Annexin-V⁺ 7-AAD⁻ as early as 2 hours after UV irradiation. Other triggers might be anti-Fas antibody or treatment with Staurosporine. Staurosporine (antibiotic AM-2282 or STS) is a natural product originally isolated from bacterium Streptomyces staurosporeus [Omura S, et al. (1977) J. Antibiot. 30 (4): 275-282].

Example 8

Apoptotic Cell Engineered to Express an Exogenous Immune Antigen

Generation of an apoptotic cell engineered to express an exogenous immune antigen is desirable for inducing a T_(reg) immune response. This immune response may be directed against a tumor-associated immune antigen as a form of tumor immunotherapy, or for inhibiting T_(H)17 immune responses directed against an exogenous antigen of microbial origin. Such apoptotic cells may be prepared, for example, as follows:

1) A cell line is transfected with the exogenous immune antigen of interest. For example, the antigen may be a tumor-associated antigen where T_(H)17 immune responses are desired against a given tumor or an immunodominant antigen derived from a bacterium or other source. This antigen can be expressed from a recombinant mammalian expression vector or viral vector engineered to encode the gene sequence for the antigen. This gene sequence can either be the full gene or a portion of it encompassing immunodominant regions if these are known. Clones derived from this transfected cell line are then propagated where they stably express the exogenous immune antigen at high levels. Alternatively, a tumor cell line is chosen that represents the tumor in the mammal. This tumor cell line would naturally express the tumor-associated antigen.

2) Apoptosis is induced in the transfected cell line or tumor cell line. The trigger chosen to induce apoptosis depends on the cell type. A skilled artisan will be able to determine the appropriate method for inducing apoptosis in a given cell line. This trigger can be UV irradiation at a dose sufficient to induce Annexin-V⁺ 7-AAD⁻ at early time points (4 hours). High doses of UV should be avoided as these induce necrosis (which does not induce T_(H)17 responses) measured by the appearance of Annexin-V⁺ 7-AAD⁺ as early as 2 hours after UV irradiation. Other triggers might be anti-Fas antibody or treatment with Staurosporine.

Example 9

Induction of a T_(H)17 Response In vivo by Transfer of T_(H)17-Inducing DC

Immunization with T_(H)17-inducing DC would be useful for inducing antigen-specific T_(H)17 responses in vivo. An example of this approach is provided for the mouse, but may be modified for immunization of humans, an example of which is also provided below. T_(H)17-inducing DC may be generated, for example, by the following steps:

1) Derivation of murine DC: Bone marrow (BM)-derived GM-CSF DC cultures are grown in RPMI medium supplemented with GM-CSF and 5% foetal bovine serum (FBS), plus 100 μg/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 nM sodium pyruvate, 1X MEM nonessential amino acids, and 2.5 μM β-mercaptoethanol (all from Sigma-Aldrich, St. Louis, Mo.), as previously described [Blander, J. M. & Medzhitov, R. (2006) Nature 440, 880-12].

2) Preparation of TLR-ligand carrying apoptotic cells expressing an exogenous immune antigen. The B cell line A20 stably expressing the exogenous immune antigen chicken ovalbumin (A20OVA) is stimulated with 25 μg/ml of the TLR ligand LPS for 4 days to generate A20OVA LPS blasts. Apoptosis of A20-OVA LPS blasts is induced by culturing cells with 0.5 μg/mL anti-CD95 (clone Jo2; BD) for four hours. Cells are then washed and counted.

3) Semi-adherent dendritic cells are harvested on ice on day 5 of GM-CSF DC cultures, and re-plated immediately in fresh GM-CSF medium at 1×10⁶ cells/well in 24-well tissue culture-treated plates. Apoptotic A20-OVA LPS blasts are added right away to the plates in the same medium and the cells were centrifuged for 2 min at 2000 rpm. DC are incubated with the phagocytic cells for a period of 4-6 hours then harvested and washed. These cells now provide the T_(H)17- inducing DC.

4) Mice are injected intravenously with 10⁷ OVA-specific T cell receptor transgenic CD4⁺ T cells called OT-II (available from the Jackson Laboratory). These OT-II T cells are introduced into the mouse as a readout for the activation of OVA-specific T_(H)17 CD4⁺ T cells. T_(H)17-inducing DC are injected intravenously into the tail veins of these mice 6 hours later.

5) Immunized mice are sacrificed on day 6. Single cell suspension are prepared from the spleens and lymph nodes of these mice. These suspensions are enriched for CD4⁺ T cells by sorting with MACS® CD4⁺ beads (Miltenyi Biotech, Auburn, Calif.) according to manufacturer's instructions. A feeder layer of irradiated syngeneic splenocytes is added to these CD4⁺ T cells, and co-cultures are immediately pulsed with various doses of OVA-derived peptide (0.1, 1, and 10 μg/ml). The peptide sequence is ISQAVHAAHAEINEAGR (SEQ ID NO: 91) and represents the immunodominant peptide derived from OVA encompassing residues 323-339 and presented by the major histocompatibility complex (MHC) class II molecule, I-A^(b). Culture supernatants are harvested at 48 hours and the levels of T_(H)17 cytokine IL-17 are measured by ELISA. Supernatants are also tested for the production of IFN-γ and IL-4 as markers of T_(H)1 and T_(H)2 responses, respectively. Production of IL-17 by these CD4 T cells will indicate the induction of an antigen-specific T_(H)17 immune response.

6) Protective immunity against tumor may be measured for example. Mice are immunized with the T_(H)17 inducing DC as in steps 1, 2, 3 and 4 above (without adoptive transfer of OT-II CD4³⁰ T cells). One week and 21 days later, mice are challenged with a small number (5×10⁴-10⁵) of tumor cells expressing OVA. These tumor cells may be an EL4 thymoma cell line engineered to express the exogenous antigen OVA, or the B cell lymphoma A20 engineered to express OVA. Tumor cells are injected subcutaneously and tumor development is monitored by measuring tumor size every 2 days over a period of 30 days. Tumor size will be measured on two perpendicular axes using a vernier caliper as described [Helmich, B. K. & Dutton, R. W. (2001) J Immunol 166, 6500-8; Taetle, R., et al. (1987) Cancer Treat Rep 71, 297-304], and tumor size will be approximated by multiplying the measured widths and lengths [Taetle, R., et al, supra ]. Mean tumor volume and standard errors of the mean will be averaged from 8-10 identically treated mice per group. Tumor development is compared between groups of immunized and unimmunized mice (8-10 mice per group, sex and age matched).

Modification for Humans:

Peripheral blood mononuclear cells (PBMCs) are enriched from apheresis by Ficoll gradient centrifugation, frozen, and stored at −180° C. as described [Palucka, A. K. et. al. (2006) J. Immunotherapy. 29:545-557]. The T_(H)17-inducing DC vaccine is generated under cGMP conditions by culturing monocytes, enriched from thawed PBMCs by 2 hours adherence, for 6 days in X-VIVO15 (BioWhittaker. Walkersville, Md.) supplemented with 1% autologous serum, GM-CSF (200 ng/mL, Leukine, Berlex Inc. (Bayer HealthCare), Montville, N.J.) and IL-4 (50 ng/mL, R&D systems) as described [Palucka et al, supra]. A third of the vaccine is loaded with control antigen keyhole limpet hemocyanin (KLH) (Biosyn Corp., Carlsbad, Calif.), and two-thrids are loaded with apoptotic TLR-ligand carrying or microbe-infected cells expressing an exogenous immune antigen. The TLR-ligand carrying or microbe-infected apoptotic cells are prepared as described in Example 7, above. The exogenous antigen may be a known immunodominant antigen derived from a bacterium, or other source, for example. Alternatively, if an immunodominant antigen is not defined, an inactivated form of, e.g., a bacterium is given to a phagocytic cell line, which is then induced to undergo apoptosis by UV irradiation (see Example 7). Following the loading procedure, DC are activated by adding tumor necrosis factor (TNF) (20 ng/mL) and soluble CD40 ligand (200 ng/mL, both from R&D Systems) in the last 30 hours of culture as described [Paleuka et al, supra]. DC are defined by CD11c surface expression, high levels of HLA-DR, CD83, and the costimulatory molecule CD80. The expression of the costimulatory molecule CD80 serves as a phenotype consistent with the activation and maturation of the T_(H)17-inducing DC.

An example of clinical monitoring can be the form of intracellular cytokine analysis for IFN-γ, IL-4 and IL-17. Frozen/thawed PBMCs are resuspended in 2×10⁶ cells/mL and 2×10⁶ PBMCs are stimulated with either the immunodominant microbial derived peptide (10 μM), the control KLH, or heat inactivated bacteria at a dose of 2-5 bacteria to 1 cell. The mAbs anti-CD28/CD49d are also added (BD bioscience) as described [Palucka et al, supra]. At 2 hours of stimulation, cells are harvested, and stained with anti-CD3 PerCP and/or anti-CD4 APC mAbs before fixation and permeabilization with BD Cytofix/Cytoperm solution (BD Pharmingen or eBioscience). The cells are then stained with anti-IFN-γ-FITC and anti-IL-17-PE (BD Pharmingen or eBioscience). PBMCs should respond to KLH as this serves as a positive control. Staining for IL-17 will indicate priming (induction) of a T_(H)17 CD4 T cell response against the exogenous immune antigen.

SUMMARY

The results described herein demonstrate that sensing by DC of infected apoptotic cells during infections triggers instructive signals critical for T_(H)17 development. They also explain the puzzling observations that some but not all microbial pathogens induce T_(H)17 cells, and indicate the importance of examining the induction of apoptosis by pathogens inducing T_(H)17 cells. Thus, T_(H)17 cells can be induced not only by the dectin-1 pathway in case of fungal infections²¹, but also by the TLR pathway in case of infected apoptotic cells. The findings of the present invention additionally show a novel role for apoptosis in host-pathogen interactions along epithelial surfaces. Thus, these data highly suggest that T_(H)17 cells are critial in mediating effector functions during bacterial infections associated with significant apoptosis and tissue damage. In addition to neutrophil and macrophage recruitment, tissue repair processes uniquely associated with the effector functions of T_(H)17 cells ^(3, 17) would aid host response against these pathogens. Since T_(H)17 cells have been correlated with autoimmune diseases¹⁻⁵, investigation of the pathways of innate recognition of infected apoptotic cells might lead to improved understanding of the causative defects in autoimmunity.

Moreover, the present invention describes invention described a previously unappreciated role of apoptosis on host-pathogen interactions across the intestinal epithelium. The association of T_(H)17 cells with autoimmune diseases, combined with a previously unrecognized apoptotic cell component for instructing T_(H)17 development in conjunction with TLR signaling, suggest breakdown in critical regulatory pathways that direct T_(H)17 specificities to microbial components rather than to self. This novel understanding will be critical for design of the next generation of therapeutic agents and vaccines aimed at treating or preventing autoimmune diseases.

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What is claimed:
 1. A method for generating a T_(H)17-inducing dendritic cell (DC) that secretes interleukin-6 (IL-6) and transforming growth factor beta (TGF-β), wherein the combined amount of IL-6 and TGF-β secreted by the T_(H)17-inducing DC is effective for inducing a T_(H)17 response when the T_(H)17-inducing DC is administered to a subject, the method comprising administering to a DC in vitro a Toll-like receptor (TLR) agonist and an apoptotic cell-associated agent in a combined amount effective for generating the T_(H)17-inducing DC.
 2. The method of claim 1, wherein the TLR agonist and the apoptotic cell-associated agent are either in direct physical association or are combined in a manner that allows internalization as a single entity by the DC in vitro.
 3. The method according to claim 1, wherein the apoptotic cell-associated agent comprises any one of the agents selected from the group consisting of an apoptotic cell, an apoptotic cell mimic, phosphatidylserine, a microbe-infected apoptotic cell, a phosphatidylserine mimic, a mimic of cell surface calreticulin translocation, and a polypeptide that is a marker of apoptosis.
 4. The method according to claim 1, wherein the TLR agonist comprises any one of the agents selected from the group consisting of a TLR ligand, a TLR ligand mimic, a synthetic or chemical TLR ligand, a cell or particle comprising a pathogen-associated molecular pattern, a microbial pathogen, a bacterium, a virus, and a viral particle.
 5. A method for generating a T_(H)17-inducing dendritic cell (DC) that secretes interleukin-6 (IL-6) and transforming growth factor beta (TGF-β), wherein the combined amount of IL-6 and TGF-β secreted by the T_(H)17-inducing DC is effective for inducing a T_(H)17 response when the T_(H)17-inducing DC is administered to a subject, the method comprising administering to a DC in vitro (i) and (ii), as a single entity or in a combined form, wherein (i) is at least one member selected from the group consisting of a Toll-like receptor (TLR) ligand, a TLR ligand mimic, a synthetic or chemical TLR ligand, a cell or particle comprising a pathogen-associated molecular pattern, a microbial pathogen, a TLR agonist, a bacterium, and a virus or viral-like particle, and wherein (ii) is at least one member selected from the group consisting of an apoptotic cell, a microbe-infected apoptotic cell, an apoptotic cell mimic, phosphatidylserine, a phosphatidylserine mimic, an apoptotic cell-associated agent, a mimic of cell surface calreticulin translocation, and a polypeptide that is a marker of apoptosis; wherein the combined amount of (i) and (ii) is effective for generating the T_(H)17-inducing DC.
 6. An immunogenic composition comprising a) a T_(H)17-inducing DC that secretes IL-6 and TGF-β, b) an immune antigen, and c) a pharmaceutically acceptable carrier or diluent, wherein a combined amount of the IL-6 and the TGF-β secreted by the T_(H)17-inducing DC and the immune antigen is effective for eliciting a T_(H)17 response to the immune antigen in a subject which is administered the immunogenic composition.
 7. The immunogenic composition of claim 6, wherein the T_(H)17-inducing DC is generated by a method comprising pre-treating a DC in vitro with a TLR agonist and an apoptotic cell-associated agent in a combined amount effective for generating the T_(H)17-inducing DC.
 8. The immunogenic composition of claim 7, wherein the TLR agonist and the apoptotic cell-associated agent are combined in a manner that allows internalization as a single entity by the DC in vitro.
 9. The immunogenic composition of claim 6, wherein the T_(H)17-inducing DC is pre-treated in vitro with the immune antigen or with a peptide fragment derived from the immune antigen.
 10. A method for inducing in a patient an antigen-specific T_(H)17-driven immune response, which comprises administering to a patient in need thereof the immunogenic composition according to claim 6 in an effective amount for inducing the antigen-specific T_(H)17-driven immune response.
 11. The method according to claim 10, wherein the antigen-specific T_(H)17-driven immune response comprises a mucosal immune response.
 12. The method according to claim 10, wherein the patient is a human.
 13. A method for treating cancer in a mammal, which comprises administering to a mammal in need thereof the immunogenic composition according to claim 6 in an effective amount for treating cancer, wherein the immune antigen in the immunogenic composition is a tumor-specific antigen.
 14. The method of claim 13, wherein the cancer is an epithelial or mixed epithelial carcinoma.
 15. The method of claim 14, wherein the epithelial or mixed epithelial carcinoma is a member selected from the group consisting of ovarian cancer, breast cancer, pancreatic cancer, lung carcinoma, laryngeal carcinoma, adenoid cystic carcinoma, epithelial carcinomas of the upper aerodigestive tract, hepatocellular carcinoma, colorectal carcinoma, lymphoepithelial carcinoma, squamous cell carcinoma, renal cell carcinoma, mixed epithelial and stromal tumors of the kidney, and renal angiomyoadenomatous tumors.
 16. The method according to claim 13, wherein the immunogenic composition is administered to the mammal by an oral or mucosal route. 